Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine

ABSTRACT

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds. The engineered ketoreductase polypeptides are optimized for catalyzing the conversion of N-methyl-3-keto-3-(2-thienyl)-1-propanamine to (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine.

This application claims the benefit, pursuant 35 U.S.C. §119(e), of U.S.Ser. No. 61/092,331, which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present disclosure relates to engineered ketoreductase polypeptidesand uses thereof for the preparation of 3-aryl-3-hydroxypropanaminesfrom the corresponding 3-aryl-3-ketopropanamines.

BACKGROUND

Enzymes belonging to the ketoreductase (KRED) or carbonyl reductaseclass (EC1.1.1.184) are useful for the synthesis of optically activealcohols from the corresponding prostereoisomeric ketone substrates andby stereospecific reduction of corresponding racemic aldehyde and ketonesubstrates. KREDs typically convert a ketone or aldehyde substrate tothe corresponding alcohol product, but may also catalyze the reversereaction, oxidation (dehydrogenation) of an alcohol substrate to thecorresponding ketone/aldehyde product. The reduction of ketones andaldehydes and the oxidation of alcohols by enzymes such as KRED requiresa co-factor, most commonly reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH),and nicotinamide adenine dinucleotide (NAD) or nicotinamide adeninedinucleotide phosphate (NADP) for the oxidation reaction. NADH and NADPHserve as electron donors, while NAD and NADP serve as electronacceptors. It is frequently observed that ketoreductases and alcoholdehydrogenases accept either the phosphorylated or thenon-phosphorylated co-factor (in its oxidized and reduced state).

KRED enzymes can be found in a wide range of bacteria and yeasts (forreviews: Kraus and Waldman, Enzyme catalysis in organic synthesis Vols.1&2.VCH Weinheim 1995; Faber, K., Biotransformations in organicchemistry, 4th Ed. Springer, Berlin Heidelberg N.Y. 2000; Hummel andKula Eur. J. Biochem. 1989 184:1-13). Several KRED gene and enzymesequences have been reported, e.g., Candida magnoliae (Genbank Acc. No.JC7338; GI:11360538) Candida parapsilosis (Genbank Acc. No. BAA24528.1;GI:2815409), Sporobolomyces salmonicolor (Genbank Acc. No. AF160799;GI:6539734).

In order to circumvent many chemical synthetic procedures for theproduction of key compounds, ketoreductases are being increasinglyemployed for the enzymatic conversion of different keto and aldehydesubstrates to chiral alcohol products. These applications can employwhole cells expressing the ketoreductase for biocatalytic reductions, orpurified enzymes in those instances where presence of multipleketoreductases in whole cells would adversely affect the stereopurityand yield of the desired product. For in vitro applications, a co-factor(NADH or NADPH) regenerating enzyme such as glucose dehydrogenase (GDH),formate dehydrogenase etc. can be used in conjunction with theketoreductase. Examples using ketoreductases to generate useful chemicalcompounds include asymmetric reduction of 4-chloroacetoacetate esters(Zhou, J. Am. Chem. Soc. 1983 105:5925-5926; Santaniello, J. Chem. Res.(S) 1984:132-133; U.S. Pat. No. 5,559,030; U.S. Pat. No. 5,700,670 andU.S. Pat. No. 5,891,685), reduction of dioxocarboxylic acids (e.g., U.S.Pat. No. 6,399,339), reduction of tert-butyl (S)chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No. 6,645,746 and WO01/40450), reduction pyrrolotriazine-based compounds (e.g., USapplication No. 2006/0286646); reduction of substituted acetophenones(e.g., U.S. Pat. No. 6,800,477); and reduction of ketothiolanes (WO2005/054491).

It is desirable to identify other ketoreductase enzymes that can be usedto carry out conversion of various keto substrates to theircorresponding chiral alcohol products.

SUMMARY

The present disclosure provides ketoreductase polypeptides having theability to reduce a 3-aryl-3-ketopropanamine to a3-aryl-3-hydroxypropanamine, polynucleotides encoding such polypeptides,and methods for making and using the polypeptides. Ketoreductasepolypeptides of the present invention are particularly useful forpreparing intermediates in the synthesis of the drug, Duloxetine (i.e.,(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine).Exemplary substrates include, for example,N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (“DMAK”; “the dimethylamino ketone substrate” or the “dimethyl substrate”) andN-methyl-3-keto-3-(2-thienyl)-1-propanamine (“MMAK”; “the monomethylamino ketone substrate” or the “monomethyl substrate”) which are reducedto (S)—N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-DMAA”;“the dimethyl amino alcohol product” or the “dimethyl product”) and(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-MMAA”; “themonomethyl amino alcohol product” or the “monomethyl product”),respectively. The ketoreductase polypeptides of the present inventionexhibit the ability to reduce the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine with particularlyhigh stereoselectivity.

In one aspect, the ketoreductase polypeptides described herein have anamino acid sequence that has one or more amino acid differences ascompared to a reference wild-type ketoreductase or an engineeredketoreductase sequence, where the differences result in an improvedproperty of the enzyme in carrying out the conversion of the specificketo substrate to the alcohol product. Generally, the engineeredketoreductase polypeptides have an improved property as compared to thenaturally-occurring wild-type ketoreductase enzymes obtained fromLactobacillus kefir (“L. kefir”; SEQ ID NO:2), Lactobacillus brevis (“L.brevis”; SEQ ID NO:4), or Lactobacillus minor (“L. minor,” SEQ IDNO:60), or compared to another engineered polypeptide, such as SEQ IDNO:6. Improvements in enzyme property can include increases in enzymeactivity, stereoselectivity, stereospecificity, thermostability, solventstability, or reduced product inhibition. In the present disclosure, theketoreductase polypeptides have, as compared to the amino acid sequenceof SEQ ID NO:2, 4 or 60, at least the following features: (1) theresidue corresponding to position 94 is an acidic residue, (2) theresidue corresponding to position 145 is an aromatic residue or leucine,and (3) the residue corresponding to position 190 is a cysteine or aconstrained residue. In some embodiments, the polypeptides of thedisclosure have, as compared to the sequences of SEQ ID NO:2, 4 and 60,at least the following features: (1) the residue corresponding toposition 94 is aspartic acid, (2) the residue corresponding to position145 is tyrosine, tryptophan, phenylalanine, or leucine, particularlyphenylalanine or leucine and (3) the residue corresponding to position190 is cysteine or proline. In some embodiments, the ketoreductasepolypeptides of the disclosure have, as compared to the sequences of SEQID NO:2, 4 or 60, at least the following features: (1) residuecorresponding to position 94 is aspartic acid, (2) residue correspondingto position 145 is phenylalanine, and (3) residue corresponding toposition 190 is proline.

In some embodiments, the engineered ketoreductases can possess a singleimproved property, or they can possess two or more improved properties,in any combination(s). For example, the engineered ketoreductasepolypeptide can have increased enzymatic activity as compared to thewild-type ketoreductase enzyme for reducing the monomethyl substrate tothe corresponding product. Improvements in enzymatic activity can bemeasured by comparing the specific activity of the ketoreductasepolypeptide with that of the wild-type ketoreductase enzyme usingstandard enzyme assays. The amount of the improvement can range from 1.5times the enzymatic activity of the corresponding wild-type or that of areference ketoreductase enzyme, to as much as 2, 5, 10, 15 times or moreimprovement of the enzymatic activity. In specific embodiments, theengineered ketoreductase enzyme exhibits improved enzymatic activitythat is at least 1.5-times, 2 times, 3 times, 4 times, 5 times, 10times, 15 times, 20 times, 25 times, 30 times, 50 times or more thanthat of the wild-type or of the reference ketoreductase enzyme.Improvements in enzyme activity also include increases instereoselectivity, stereospecificity, thermostability, solventstability, or reduced product inhibition.

In some embodiments, the ketoreductase polypeptides herein are improvedas compared to SEQ ID NO:2 or SEQ ID NO:6 with respect to their rate ofenzymatic activity, i.e., their rate of converting the monomethylsubstrate to the corresponding product. In some embodiments, theketoreductase polypeptides are capable of converting the monomethylsubstrate to product at a rate that is at least 1.5 times, 2 times, 5times, 10 times, or 15 times the rate displayed by the ketoreductase ofSEQ ID NO:2 or SEQ ID NO:6.

In some embodiments, the ketoreductase polypeptides are capable ofconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine at a rate that isimproved over a reference polypeptide having the amino acid sequence ofSEQ ID NO:6. In some embodiments, such ketoreductase polypeptides arealso capable of converting the monomethyl substrate to product with apercent stereomeric excess of at least about 95%. In some embodiments,such ketoreductase polypeptides are also capable of converting themonomethyl substrate to product with a percent stereomeric excess of atleast about 99%. Exemplary polypeptides with such properties include,but are not limited to, polypeptides which comprise amino acid sequencescorresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, and 32. Because the reference polypeptide having the amino acidsequence of SEQ ID NO:6 is capable of converting the monomethylsubstrate to product at a rate improved over that of the wild-typeenzyme of SEQ ID NO:2, the polypeptides herein that are improved overSEQ ID NO:6 are also improved over wild-type.

In some embodiments, the ketoreductase polypeptides of the disclosureare capable of converting the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with a percentstereomeric excess of at least about 99% and at a rate that is at least5-10 times the rate of a reference polypeptide having the amino acidsequence of SEQ ID NO:6. Exemplary polypeptides with such propertiesinclude, but are not limited to, polypeptides which comprise an aminoacid sequence corresponding to SEQ ID NO: 8, 10, and 20.

In some embodiments, the ketoreductase polypeptides of the disclosureare capable of converting the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with a percentstereomeric excess of at least about 99% and at a rate that is at least10-15 times the rate of a reference polypeptide having the amino acidsequence of SEQ ID NO:6. Exemplary polypeptides with such propertiesinclude, but are not limited to, polypeptides which comprise an aminoacid sequence corresponding to SEQ ID NO: 12, 14, 16, 18, and 22.

In some embodiments, the ketoreductase polypeptides are capable ofconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine tothe product (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with apercent stereomeric excess of at least about 99% and at a rate that isat least 15 times the rate of a reference polypeptide having the aminoacid sequence of SEQ ID NO:6. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides which compriseamino acid sequences corresponding to SEQ ID NO: 24, 26, 28, 30, and 32.

In some embodiments, the ketoreductase polypeptides of the disclosureare capable of converting at least about 95% of the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine in less than about 24hours when carried out with greater than about 100 g/L of substrate andless than about 5 g/L of the polypeptide. Exemplary polypeptides thathave this capability include, but are not limited to, polypeptides whichcomprise amino acid sequences corresponding to SEQ ID NO: 24, 26, 28,30, and 32.

In some embodiments, the ketoreductase polypeptides can highlystereoselectively reduce the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine in greater than about99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%stereomeric excess. Exemplary ketoreductase polypeptides with such highstereoselectivity include, but are not limited to, the polypeptidescomprising the amino acid sequences corresponding to SEQ ID NO: 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

In some embodiments, an improved ketoreductase polypeptide comprises anamino acid sequence that corresponds to the sequence formula provided inSEQ ID NO:61 or SEQ ID NO:62 or SEQ ID NO:63, or a region thereof, suchas residues 90-211. SEQ ID NO:61 is based on the wild-type amino acidsequence of the Lactobacillus kefir ketoreductase (SEQ ID NO:2); SEQ IDNO:62 is based on the wild-type amino acid sequence of the Lactobacillusbrevis ketoreductase (SEQ ID NO:4); and SEQ ID NO:63 is based on thewild-type amino acid sequence of the Lactobacillus minor ketoreductase(SEQ ID NO:60). SEQ ID NOs:61, 62 and 63 specify that (1) residue 94 isan acidic amino acid, (2) residue 145 is an aromatic amino acid orleucine, and (3) residue 190 is a cysteine or a constrained residue.

In some embodiments, an improved ketoreductase polypeptide of thedisclosure is based on the sequence formula of SEQ ID NO:61, 62 or 63and can comprise an amino acid sequence that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to reference sequence based on SEQ ID NO:2, 4 or 60, with theproviso that (1) the amino acid residue corresponding to position 94 isan acidic amino acid, (2) the amino acid residue corresponding toposition 145 is aromatic amino acid or leucine, and (3) the amino acidresidue corresponding to position 190 is cysteine or proline, whereinthe ketoreductase polypeptides have at least the specified provisos. Insome embodiments, the ketoreductase polypeptides have the specifiedsequence identity to a reference sequence based on SEQ ID NO:2, 4, or60, or a region there of, such as residues 90-211, with the proviso that(1) the amino acid residue corresponding to position 94 is asparticacid, (2) the amino acid residue corresponding to position 145 istyrosine, phenylalanine or leucine, particularly phenylalanine orleucine, and (3) the amino acid residue corresponding to position 190 iscysteine or proline, wherein the ketoreductase polypeptides have atleast the specified provisos. In some embodiments, the ketoreductasepolypeptides have the specified sequence identity to a referencesequence based on SEQ ID NO:2, 4, or 60, or a region there of, such asresidues 90-211, with the proviso that (1) the amino acid residuecorresponding to position 94 is aspartic acid, (2) the amino acidresidue corresponding to position 145 is phenylalanine or leucine, and(3) the amino acid residue corresponding to position 190 is proline,wherein the ketoreductase polypeptides have the specified provisos. Insome embodiments, the ketoreductase polypeptides can additionally haveone or more amino acid residue differences as compared to the referencesequences above. These differences can be amino acid insertions,deletions, substitutions, or any combination of such changes. In someembodiments, the amino acid sequence differences can comprisenon-conservative, conservative, as well as a combination ofnon-conservative and conservative amino acid substitutions. Variousamino acid residue positions where such changes can be made aredescribed herein.

In some embodiments, an improved ketoreductase polypeptide of thedisclosure is based on the sequence formula of SEQ ID NO:89, 90 or 91and can comprise a region or domain that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a region or domain thereof, such as residues 90-211 ofreference sequence based on SEQ ID NO:2, 4 or 60, with the proviso that(1) the amino acid residue corresponding to position 94 is an acidicamino acid, (2) the amino acid residue corresponding to position 145 isaromatic amino acid or leucine, and (3) the amino acid residuecorresponding to position 190 is cysteine or proline, wherein theketoreductase polypeptides have at least the specified provisos. In someembodiments, the ketoreductase polypeptides have the specified sequenceidentity to a reference sequence based on SEQ ID NO:2, 4, or 60, or aregion there of, such as residues 90-211, with the proviso that (1) theamino acid residue corresponding to position 94 is aspartic acid, (2)the amino acid residue corresponding to position 145 is tyrosine,phenylalanine or leucine, and (3) the amino acid residue correspondingto position 190 is cysteine or proline, and wherein the ketoreductasepolypeptides have at least the specified provisos. In some embodiments,the ketoreductase polypeptides have the specified sequence identity to areference sequence based on SEQ ID NO:2, 4, or 60, or a region there of,such as residues 90-211, with the proviso that (1) the amino acidresidue corresponding to position 94 is aspartic acid, (2) the aminoacid residue corresponding to position 145 is phenylalanine, and (3) theamino acid residue corresponding to position 190 is proline, and whereinthe ketoreductase polypeptides have at least the specified provisos. Insome embodiments, the ketoreductase polypeptides can additionally haveone or more amino acid residue differences in the defined region. Insome embodiments, the amino acid sequence differences can comprisenon-conservative, conservative, as well as a combination ofnon-conservative and conservative amino acid substitutions. Variousamino acid residue positions where such changes can be made aredescribed herein.

In some embodiments, an improved ketoreductase comprises an amino acidsequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence corresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, and 32, wherein the improved ketoreductase polypeptide aminoacid sequence includes any one set of the specified amino acidsubstitution combinations presented in Table 2. In some embodiments,these ketoreductase polypeptides can have from about 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, or about 1-35 mutations (i.e., differences) atother amino acid residues. In some embodiments, the number of mutationscan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22,24, 26, 30, 35 or about 40 amino acid residues. In some embodiments, themutations are conservative mutations.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductases described herein orpolynucleotides that hybridize to such polynucleotides under highlystringent conditions. The polynucleotide can include promoters and otherregulatory elements useful for expression of the encoded engineeredketoreductase, and can utilize codons optimized for specific desiredexpression systems. Exemplary polynucleotides include, but are notlimited to, SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and31. Exemplary polynucleotides also include polynucleotides encodingpolypeptides that correspond to the sequence formula of SEQ ID NO:61, 62or 63.

In another aspect, the present disclosure provides host cells comprisingthe polynucleotides and/or expression vectors described herein. The hostcells may be L. kefir or L. brevis or L. minor, or they may be adifferent organism, such as E. coli. The host cells can be used for theexpression and isolation of the engineered ketoreductase enzymesdescribed herein, or, alternatively, they can be used directly for theconversion of the substrate to the stereoisomeric product.

Whether carrying out the method with whole cells, cell extracts orpurified ketoreductase enzymes, a single ketoreductase enzyme may beused or, alternatively, mixtures of two or more ketoreductase enzymesmay be used.

The ketoreductase enzymes described herein are useful for catalyzing thereduction reaction of the keto group in the 3-aryl-3-ketopropanaminehaving the structural formula (I):

to the corresponding (S)-3-aryl-3-hydroxypropanamine having thestructural formula (II):

wherein for (I) and (II), R₁ and R₂ are each independently selected fromthe group consisting of hydrogen, an optionally substituted lower alkyl,an optionally substituted cycloalkyl, an optionally substituted aryl, oralternatively, where R₁ and R₂ together form an optionally substitutedcycloalkyl or an optionally substituted cycloaryl having 3-7 carbonatoms; R₃, R₄, R₅, and R₆ are each independently selected from the groupconsisting of hydrogen and an optionally substituted lower alkyl; and R₇is an optionally substituted aryl.

The ketoreductase polypeptides described herein are particularly capableof reducing the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine(III):

to the corresponding stereoisomeric alcohol product of structuralformula (II), (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“themonomethyl product”):

with high stereoselectivity.

In some embodiments, the invention provides a method for reducing thesubstrate having the chemical formula (I) to the corresponding productof structural formula (II), comprising contacting or incubating thesubstrate with a ketoreductase polypeptide described herein underreaction conditions suitable for reducing or converting the substrate tothe product. For example, in some embodiments of this method, theketoreductase polypeptides have, as compared to the L. kefir or L.brevis or L. minor KRED sequences of SEQ ID NO: 2, 4 or 60, at least thefollowing features: (1) residue corresponding to X94 is an acidic aminoacid, (2) residue corresponding to X145 is an aromatic amino acid orleucine, and (3) residue corresponding to X190 is a cysteine or aconstrained amino acid. In some embodiments of this method, theketoreductase polypeptide used in the method can have, as compared tothe wild-type L. kefir or L. brevis or L. minor KRED sequences of SEQ IDNO: 2, 4 and 60, respectively, at least the following features: (1)residue corresponding to residue X94 is glycine, (2) residuecorresponding to position 145 tyrosine, tryptophan, phenylalanine, orleucine, particularly phenylalanine or leucine, and (3) residuecorresponding to X190 is cysteine or proline, particularly proline. Insome embodiments of this method, the ketoreductase polypeptides have, ascompared to the L. kefir or L. brevis or L. minor KRED sequences of SEQID NO: 2, 4 or 60, the following features: (1) residue corresponding toX94 is glycine, (2) residue corresponding to X145 phenylalanine and (3)residue corresponding to X190 is proline.

In some embodiments of this method for reducing the substrate to theproduct, the monomethyl substrate (III) is reduced to the monomethylproduct (IV) in greater than about 99% stereomeric excess, wherein theketoreductase polypeptide comprises a sequence that corresponds to SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

In some embodiments of this method for reducing the monomethly substrate(III) to the monomethyl product (IV), at least about 95% of thesubstrate is converted to the product in less than about 24 hours whencarried out with greater than about 100 g/L of substrate and less thanabout 5 g/L of the polypeptide, wherein the polypeptide comprises anamino acid sequence corresponding to SEQ ID NO: 24, 26, 28, 30, or 32.

In some embodiments of this method for reducing the monomethyl substrate(III) to the monomethyl product (IV), at least about 10-20% of 1 g/Lsubstrate is converted to the product in less than about 24 hours withabout 10 g/L of the polypeptide, wherein the polypeptide comprises anamino acid sequence corresponding to 12, 14, 16, 18, 22, 24, 26, 28, 30,or 32.

In some embodiments, the method for reducing or converting the compoundN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine is carried out at apH of from about 5 to about 8.0. In some embodiments, the contacting iscarried out a pH of about 6.5.

In some embodiments, the methods relate to the use of ketoreductasepolypeptides of the present invention in the synthesis of an(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine, the method comprising:

(a) providing a 3-aryl-3-ketopropanamine substrate having the structureof formula (I):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl;(b) contacting the 3-aryl-3-ketopropanamine substrate with one or moreketoreductase polypeptides of the present invention in a reactionmixture under conditions suitable for reduction or conversion of thesubstrate to an (S)-3-aryl-3-hydroxypropanime product having thestructural formula (II):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and a anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl;(c) demethylating the (S)-3-aryl-3-hydroxypropanamine (i.e.,N,N-dimethyl-3-hydroxy-3-(aryl)-propanamine) product of step (b) in areaction mixture under conditions suitable for producing an(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine having the formula ofstructure (II):

wherein one of R₁ and R₂ is methyl and the other is hydrogen, R₃, R₄,R₅, and R₆ are each independently selected from the group consisting ofhydrogen and an optionally substituted lower alkyl, and R₇ is anoptionally substituted aryl.

In some embodiments, the methods relate to the use of the ketoreductasepolypeptides of the present invention in the synthesis of a3-aryloxy-3-arylpropanamine, the method comprising:

(a) providing a 3-aryl-3-ketopropanamine having the structure of formula(I):

(b) contacting the 3-aryl-3-ketopropanamine with a ketoreductasepolypeptide of the present invention in a reaction mixture underconditions sufficient to produce an (S)-3-aryl-3-hydroxypropanaminehaving the structure of formula (II):

and(c) contacting the (S)-3-aryl-3-hydroxypropanamine with an activatedaryl compound in a reaction mixture under conditions sufficient toproduce the (S)-3-aryloxy-3-(aryl)-propanamine having the structure offormula (VII):

wherein for (I), (II), and (VII), R₁ and R₂ are each independentlyselected from the group consisting of hydrogen, an optionallysubstituted lower alkyl, an optionally substituted cycloalkyl, anoptionally substituted aryl, or alternatively, where R₁ and R₂ togetherform an optionally substituted cycloalkyl or an optionally substitutedcycloaryl having 3-7 carbon atoms; R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl; and R₇ is an optionally substitutedaryl and additionally, for (VII), Ar is an optionally substituted arylgroup.

In some embodiments, the methods relate to use of the ketoreductasepolypeptides in the synthesis of Duloxetine,(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine,having the structure of formula (VIII):

including salts, hydrates and solvates thereof. Thus, in a method forthe synthesis of the compound of formula (VIII), a step in the methodcomprises reducing the substrate of structural formula (III) to thecorresponding product of formula (IV) by contacting or incubating thesubstrate with a ketoreductase polypeptide described herein, therebyreducing the substrate to the product compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the role of ketoreductases in the conversion of thecompound of formula (III), N-methyl-3-keto-3-(2-thienyl)-1-propanamine,to the corresponding chiral alcohol product of formula (IV),(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine. In the illustratedreaction, the reduction uses a cofactor such as NADPH. A glucosedehydrogenase can be used to convert/recycle the NADP+ to NADPH. Glucoseis converted to gluconic acid, which is in turn converted to its sodiumsalt (sodium gluconate) with the addition of sodium hydroxide.

DETAILED DESCRIPTION Definitions

As used herein, the following terms are intended to have the followingmeanings

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide having an enzymatic capability of reducing a carbonyl groupto its corresponding alcohol. More specifically, the ketoreductasepolypeptides of the invention are capable of stereoselectively reducingthe compound of formula (I) to the corresponding product of formula(II). The polypeptide typically utilizes a cofactor reduced nicotinamideadenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotidephosphate (NADPH) as the reducing agent. Ketoreductases as used hereininclude naturally occurring (wild type) ketoreductases as well asnon-naturally occurring engineered polypeptides generated by humanmanipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, by the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WisconsinSoftware Package), or by visual inspection (see generally, CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)), all of whichare incorporated herein by reference. Examples of algorithms that aresuitable for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul etal., 1977, Nucleic Acids Res. 3389-3402, respectively, which areincorporated herein by reference. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation website. This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al., supra, which is incorporated herein byreference). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915, which is incorporated herein by reference). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a “comparison window” to identify and compare local regions ofsequence similarity.

In some embodiments, a “reference sequence” can be based on a primaryamino acid sequence, where the reference sequence is a sequence that canhave one or more changes in the primary sequence. For instance, areference sequence “based on SEQ ID NO:4 having at the residuecorresponding to X94 an aspartic acid” refers to a reference sequence inwhich the corresponding residue at X94 in SEQ ID NO:4 has been changedto an aspartic acid.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredketoreductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned. In someembodiments, unless specifically described otherwise, the terms “Xn”,“residue position n”, or “residue n”, where “n” is the number positionof a residue with respect to a reference sequence, are to be used in thecontext of “corresponding to” as described herein.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diasteromers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a ketoreductase polypeptide that iscapable of converting or reducing the substrate to the corresponding(5)-product with at least about 99% stereomeric excess.

“Stereospecificity” refers to the preferential conversion in a chemicalor enzymatic reaction of one stereoisomer over another.Stereospecificity can be partial, where the conversion of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is converted.

“Chemoselectivity” refers to the preferential formation in a chemical orenzymatic reaction of one product over another.

“Improved enzyme property” refers to a ketoreductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference ketoreductase. For the engineered ketoreductase polypeptidesdescribed herein, the comparison is generally made to the wild-typeketoreductase enzyme, although in some embodiments, the referenceketoreductase can be another improved engineered ketoreductase. Enzymeproperties for which improvement is desirable include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate), thermal stability, pH activityprofile, cofactor requirements, refractoriness to inhibitors (e.g.,product inhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered ketoreductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of KRED) ascompared to the reference ketoreductase enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 100% improved over the enzymatic activity of thecorresponding wild-type ketoreductase enzyme, to as much as 200%, 500%,1000%, 1500% or more over the enzymatic activity of the naturallyoccurring ketoreductase or another engineered ketoreductase from whichthe ketoreductase polypeptides were derived. In specific embodiments,the engineered ketoreductase enzyme exhibits improved enzymatic activityin the range of a 100% to 200%, 200% to 1000%, up to and more than a1500% improvement over that of the parent, wild-type or other referenceketoreductase enzyme. It is understood by the skilled artisan that theactivity of any enzyme is diffusion limited such that the catalyticturnover rate cannot exceed the diffusion rate of the substrate,including any required cofactors. The theoretical maximum of thediffusion limit, or k_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹s⁻¹). Hence, any improvements in the enzyme activity of theketoreductase will have an upper limit related to the diffusion rate ofthe substrates acted on by the ketoreductase enzyme. Ketoreductaseactivity can be measured by any one of standard assays used formeasuring ketoreductase, such as a decrease in absorbance orfluorescence of NADPH due to its oxidation with the concomitantreduction of a ketone to an alcohol, or by product produced in a coupledassay. Comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic reduction of the substrate to thecorresponding product. “Percent conversion” refers to the percent of thesubstrate that is reduced to the product within a period of time underspecified conditions. Thus, the “enzymatic activity” or “activity” of aketoreductase polypeptide can be expressed as “percent conversion” ofthe substrate to the product.

“Thermostable” refers to a ketoreductase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g. 40-80° C.) for a period of time (e.g. 0.5-24hrs) compared to the untreated enzyme.

“Solvent stable” refers to a ketoreductase polypeptide that maintainssimilar activity (more than e.g. 60% to 80%) after exposure to varyingconcentrations (e.g, 0.5-99%) of solvent (isopropyl alcohol,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, etc.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a ketoreductase polypeptide that maintains similaractivity (more than e.g. 60% to 80%) after exposure to high or low pH(e.g. 8 to 12 or 4.5-6) for a period of time (e.g. 0.5-24 hrs) comparedto the untreated enzyme.

“Thermo- and solvent stable” refers to a ketoreductase polypeptide thatare both thermostable and solvent stable.

“Derived from” as used herein in the context of engineered ketoreductaseenzymes, identifies the originating ketoreductase enzyme, and/or thegene encoding such ketoreductase enzyme, upon which the engineering wasbased. For example, the engineered ketoreductase enzyme of SEQ ID NO:8was obtained by artificially evolving, over multiple generations thegene encoding the Lactobacillus kefir ketoreductase enzyme of SEQ IDNO:2. Thus, this engineered ketoreductase enzyme is “derived from” thewild-type ketoreductase of SEQ ID NO: 2.

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (O), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (O),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-pro (P) and L-his (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine.” The amino acid L-Cysteine (C) is unusual in that it can formdisulfide bridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. Table 1 below shows exemplary conservative substitutions.

TABLE 1 Conservative Substitutions Residue Possible ConservativeMutations A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L,V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic(D, E) K, R Other basic (K, R) P, H Other constrained (P, H) N, Q, S, TOther polar (N, Q, S, T) Y, W, F Other aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 3 ormore amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 7 or more amino acids, 8 or more amino acids, 10 ormore amino acids, 12 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered ketoreductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered ketoreductase enzymes comprise insertions of oneor more amino acids to the naturally occurring ketoreductase polypeptideas well as insertions of one or more amino acids to other improvedketoreductase polypeptides. Insertions can be in the internal portionsof the polypeptide, or to the carboxy or amino terminus. Insertions asused herein include fusion proteins as is known in the art. Theinsertion can be a contiguous segment of amino acids or separated by oneor more of the amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of a full-length ketoreductase polypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved ketoreductase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved ketoreductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure ketoreductase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved ketoreductases polypeptide is a substantially pure polypeptidecomposition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl.Acad. Sci. USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846;Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991,Nucleic Acids Res 19:698); Sambrook et al., supra); Suggs et al., 1981,In Developmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit. Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered ketoreductase enzyme of the present disclosure.

“Hybridization stringency” relates to such washing conditions of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theketoreductases enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariat analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29, which are incorporatedherein by reference). Codon usage tables are available for a growinglist of organisms (see for example, Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292;Duret, et al., supra; Henaut and Danchin, “Escherichia coli andSalmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C.,p. 2047-2066, which are incorporated herein by reference). The datasource for obtaining codon usage may rely on any available nucleotidesequence capable of coding for a protein. These data sets includenucleic acid sequences actually known to encode expressed proteins(e.g., complete protein coding sequences-CDS), expressed sequence tags(ESTS), or predicted coding regions of genomic sequences (see forexample, Mount, D., Bioinformatics: Sequence and Genome Analysis,Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281;Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270, all of which areincorporated herein by reference).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide of thepresent disclosure. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polynucleotide and/or polypeptide.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of the coding region. The control sequence maycomprise an appropriate promoter sequence. The promoter sequencecontains transcriptional control sequences, which mediate the expressionof the polypeptide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

“Cofactor regeneration system” refers to a set of reactants thatparticipate in a reaction that reduces the oxidized form of the cofactor(e.g., NADP+ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD+ or NADP+, respectively, are known in the art and may be usedin the methods described herein.

Ketoreductase Peptides

The present disclosure provides engineered ketoreductase (“KRED”)enzymes that are useful for reducing or converting a3-aryl-3-ketopropanamine substrate having the structural formula (I):

to the corresponding (S)-3-aryl-3-hydroxypropanamine having thestructural formula (II):

wherein for (I) and (II), R₁ and R₂ are each independently selected fromthe group consisting of hydrogen, an optionally substituted lower alkyl,an optionally substituted cycloalkyl, an optionally substituted aryl, oralternatively, where R₁ and R₂ together form an optionally substitutedcycloalkyl or an optionally substituted cycloaryl having 3-7 carbonatoms; R₃, R₄, R₅, and R₆ are each independently selected from the groupconsisting of hydrogen and an optionally substituted lower alkyl; and R₇is an optionally substituted aryl.

As described above, suitable substrates for the ketoreductasepolypeptides of the present invention include those having thesubstituents described for formula (I) which may be optionallysubstituted. The term “optionally substituted” refers to the replacementof hydrogen with a monovalent or divalent radical. Suitable substitutiongroups include, for example, hydroxyl, nitro, amino, imino, cyano, halo,thio, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino,imidino, guanidine, sulfonamide, carboxyl, formyl, lower alkyl, loweralkoxy, and the like. Products having the structure of formula (II) willhave the same optionally substituted substituents described above.

As used herein, the term “lower alkyl” refers to branched or straightchain alkyl groups comprising one to ten carbon atoms that areunsubstituted or substituted, e.g., with one or more halogen, hydroxyl,and the like. The term “lower alkoxy” refers herein to RO—, wherein R islower alkyl. Representative examples of lower alkoxy groups includemethoxy, ethoxy, t-butoxy, trifluoromethoxy, and the like.

“Aryl” refers herein to monocyclic and polycyclic aromatic groups havingfrom 3 to 14 backbone carbon or hetero atoms, and includes bothcarbocyclic aryl groups and heterocyclic aryl groups. Carbocyclic arylgroups are aryl groups in which all ring atoms in the aromatic ring arecarbon. The term “heterocyclic aryl” refers herein to aryl groups havingfrom 1 to 4 heteroatoms as ring atoms in an aromatic ring with theremainder of the ring atoms being carbon atoms. When used in connectionwith aryl substituents, the term “polycyclic” refers herein to fused andnon-fused cyclic structures in which at least one cyclic structure isaromatic. Exemplary aryl moieties employed as substituents in compoundsof the present invention include phenyl, naphthyl, thiophenyl, and thelike. Exemplary substituted aryl substituents include benzyl, and thelike.

“Cycloalkyl” refers herein to a mono- or polycylic, heterocyclic orcarbocyclic alkyl substituent. Typical cycloalkyl substituents have from3 to 10 backbone (i.e., ring) atoms in which each backbone atom iseither a carbon or a heteroatom. Suitable heteroatoms include nitrogen,oxygen, and sulfur.

“Cycloaryl” refers herein to a mono- or polycyclic, heterocyclic orcarbocyclic alkyl substituent. Typical cycloaryl substituents have from3 to 10 backbone atoms in which each backbone atom is either a carbon ora heteroatom. Suitable heteroatoms include nitrogen, oxygen, and sulfur.

Typical substrates have the structure of formula (I) where R₁ and R₂ areeach independently hydrogen, a lower alkyl of from one to ten or fromone to six carbon atoms, or a phenyl. Usually, one of R₁ and R₂ ishydrogen and the other is methyl or both R₁ and R₂ are methyl. Each ofR₃, R₄, R₅, and R₆ is typically hydrogen or a lower alkyl of from one toten or from one to six carbon atoms, and more typically each of R₃, R₄,R₅, and R₆ is hydrogen. R₇ is typically an optionally substitutedthiophenyl (such as, for example, an optionally substituted 2-thienyl oran optionally substituted 3-thienyl) or an optionally substitutedphenyl. More typically, R₇ is 2-thienyl or phenyl. Useful substrateshave the combination of substituents: R₁ is hydrogen, R₂ is methyl,R₃-R₆ are each hydrogen, R₇ is selected from the group consisting of2-thienyl and phenyl; and R₁ and R₂ are methyl, R₃-R₆ are each hydrogen,R₇ is selected from the group consisting of 2-thienyl and phenyl.Reduction of these substrates yields a corresponding3-aryl-3-hydroxypropanamine product having the structure of formula (II)with R₁-R₇ as described above.

Ketoreductase polypeptides of the present invention are particularlyuseful for stereoselectively reducing or converting the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine (“the monomethyl substrate”,i.e., a substrate compound having structural formula (I) where one or R₁and R₂ is hydrogen and the other is methyl, R₃-R₆ are each hydrogen, andR₇ is 2-thienyl) to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“the monomethylproduct”, i.e., a substrate compound having structural formula (II)where one of R₁ and R₂ is hydrogen and the other is methyl, R₃-R₆ areeach hydrogen, and R₇ is 2-thienyl) and having an improved property whencompared with the naturally-occurring, wild-type KRED enzyme obtainedfrom L. kefir (SEQ ID NO:2) or L. brevis (SEQ ID NO:4) or L. minor (SEQID NO:60) or when compared with another engineered ketoreductase enzyme.Enzyme properties for which improvement is desirable include, but arenot limited to, enzymatic activity, thermal stability, pH activityprofile, cofactor requirements, refractoriness to inhibitors (e.g.,product inhibition), stereospecificity, stereoselectivity, and solventstability. The improvements can relate to a single enzyme property, suchas enzymatic activity, or a combination of different enzyme properties,such as enzymatic activity and stereoselectivity.

For the ketoreductase polypeptides described herein, the amino acidsequence of the ketoreductases have, as compared to the referencesequence of SEQ ID NO:2, 4, or 60, the requirement that: (1) residuecorresponding to residue position 94 is an acidic residue, (2) residuecorresponding to residue position 145 is an aromatic residue or leucine,and (3) residue corresponding to residue position 190 is a cysteine or aconstrained residue. In some embodiments, the ketoreductase polypeptideshave, as compared to the KRED sequences of SEQ ID NO:2, 4 or 60, thefollowing features: (1) residue corresponding to residue position 94 isaspartic acid, (2) residue corresponding to residue position 145 isphenylalanine, tyrosine, tryptophan or leucine, and (3) residuecorresponding to residue position 190 is cysteine or proline. In someembodiments, the ketoreductase polypeptides have, as compared to theKRED sequences of SEQ ID NO:2, 4 or 60, the following features: (1)residue corresponding to residue position 94 is aspartic acid, (2)residue corresponding to residue position 145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine, and (3)residue corresponding to residue position 190 is cysteine or proline,particularly proline. In some embodiments, the ketoreductasepolypeptides have, as compared to the KRED sequences of SEQ ID NO:2, 4or 60, the following features: (1) residue corresponding to residueposition 94 is aspartic acid, (2) residue corresponding to residueposition 145 is phenylalanine, and (3) residue corresponding to residueposition 190 is proline.

In some embodiments, as noted above, the engineered ketoreductase withimproved enzyme activity is described with reference to Lactobacilluskefir ketoreductase of SEQ ID NO:2, Lactobacillus brevis ketoreductaseof SEQ ID NO:4, Lactobacillus minor of SEQ ID NO:60, or anotherengineered ketoreductase, such as SEQ ID NO:6. The amino acid residueposition is determined in these ketoreductases beginning from theinitiating methionine (M) residue (i.e., M represents residue position1), although it will be understood by the skilled artisan that thisinitiating methionine residue may be removed by biological processingmachinery, such as in a host cell or in vitro translation system, togenerate a mature protein lacking the initiating methionine residue. Theamino acid residue position at which a particular amino acid or aminoacid change is present in an amino acid sequence is sometimes describeherein in terms “Xn”, or “position n”, where n refers to the residueposition. Where the amino acid residues at the same residue positiondiffer between the ketoreductases, the different residues are denoted byan “/” with the arrangement being, for example, “kefir residue/brevisresidue/minor.” A substitution mutation, which is a replacement of anamino acid residue in a corresponding residue of a reference sequence,for example the wildtype ketoreductases of SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:60 with a different amino acid residue is denoted by thesymbol “→”.

Herein, mutations are sometimes described as a mutation “to a” type ofamino acid. For example, residue 211 can be mutated “to a” basicresidue. But the use of the phrase “to a” does not exclude mutationsfrom one amino acid of a class to another amino acid of the same class.For example, residue 211 can be mutated from a lysine to an arginine.

The naturally occurring polynucleotide encoding the naturally occurringketoreductase of Lactobacillus kefir, Lactobacillus brevis, orLactobacillus minor (also referred to as “ADH” or “alcoholdehydrogenase”) can be obtained from the isolated polynucleotide knownto encode the ketoreductase activity (e.g., Genbank accession no.AAP94029 GI:33112056 or SEQ ID NO:3 for Lactobacillus kefir; Genbankaccession no. CAD66648 GI:28400789 or SEQ ID NO:1 for Lactobacillusbrevis; and U.S. Pat. Appl. No. 20040265978 or SEQ ID NO:60 forLactobacillus minor).

In some embodiments, the ketoreductase polypeptides herein can have anumber of modifications to the reference sequence (e.g., an engineeredketoreductase polypeptide) to result in an improved ketoreductase enzymeproperty. In such embodiments, the number of modifications to the aminoacid sequence can comprise one or more amino acids, 2 or more aminoacids, 3 or more amino acids, 4 or more amino acids, 5 or more aminoacids, 6 or more amino acids, 8 or more amino acids, 10 or more aminoacids, 15 or more amino acids, or 20 or more amino acids, up to 10% ofthe total number of amino acids, up to 10% of the total number of aminoacids, up to 10% of the total number of amino acids; up to 20% of thetotal number of amino acids, or up to 30% of the total number of aminoacids of the reference polypeptide sequence. In some embodiments, thenumber of modifications to the naturally occurring polypeptide or anengineered polypeptide that produces an improved ketoreductase propertymay comprise from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35or about 1-40 modifications of the reference sequence. In someembodiments, the number of modifications can be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 aminoacid residues. The modifications can comprise insertions, deletions,substitutions, or combinations thereof.

In some embodiments, the modifications comprise amino acid substitutionsto the reference sequence. Substitutions that can produce an improvedketoreductase property may be at one or more amino acids, 2 or moreamino acids, 3 or more amino acids, 4 or more amino acids, 5 or moreamino acids, 6 or more amino acids, 8 or more amino acids, 10 or moreamino acids, 15 or more amino acids, or 20 or more amino acids, up to10% of the total number of amino acids, up to 10% of the total number ofamino acids, up to 20% of the total number of amino acids, or up to 30%of the total number of amino acids of the reference enzyme sequence. Insome embodiments, the number of substitutions to the naturally occurringpolypeptide or an engineered polypeptide that produces an improvedketoreductase property can comprise from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35 or about 1-40 amino acid substitutions of thereference sequence. In some embodiments, the number of substitutions canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24,26, 30, 35 or about 40 substitutions at other amino acid residues.

In some embodiments, the improved property (as compared to wild-type oranother engineered polypeptide) of the ketoreductase polypeptide is withrespect to an increase of its stereoselectivity for reducing orconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine tothe product (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine. In someembodiments, the improved property of the ketoreductase property is withrespect to an increase in stereoselectivity, i.e., herein, an increasein the stereomeric excess of the product. In some embodiments, theimproved property of the ketoreductase polypeptide is with respect to anincrease in its ability to convert or reduce a greater percentage of thesubstrate to the product. In some embodiments, the improved property ofthe ketoreductase polypeptide is with respect to an increase in its rateof conversion of the substrate to the product. This improvement inenzymatic activity can be manifested by the ability to use less of theimproved polypeptide as compared to the wild-type or other referencesequence (for example, SEQ ID NO:6) to reduce or convert the same amountof product. In some embodiments, the improved property of theketoreductase polypeptide is with respect to its stability orthermostability. In some embodiments, the ketoreductase polypeptide hasmore than one improved property.

In some embodiments, the ketoreductase polypeptide of the disclosure iscapable of converting the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine to the product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“the monomethylproduct”), with a percent stereomeric excess of at least about 99% andat a rate that is improved over a reference polypeptide having the aminoacid sequence of SEQ ID NO:6. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides which compriseamino acid sequences corresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, and 32. Because the reference polypeptide havingthe amino acid sequence of SEQ ID NO:6 is capable of converting themonomethyl substrate to the corresponding monomethyl product at a rategreater than that of wild-type (e.g., SEQ ID NO:2), the polypeptidesherein that are improved over SEQ ID NO:6 are also improved overwild-type.

In some embodiments, the ketoreductase polypeptide is capable ofconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine tothe product (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with apercent stereomeric excess of at least about 99% and at a rate that isat least 5-10 times greater than a reference polypeptide having theamino acid sequence of SEQ ID NO:6. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides which compriseamino acid sequences corresponding to SEQ ID NO: 8, 10, and 20.

In some embodiments, the ketoreductase polypeptide is capable ofconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine tothe product (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with apercent stereomeric excess of at least about 99% and at a rate that isat least 10-15 times greater than a reference polypeptide having theamino acid sequence of SEQ ID NO:6. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides which compriseamino acid sequences corresponding to SEQ ID NO: 12, 14, 16, 18, 22, 24,26, 28, 30, and 32.

In some embodiments, the ketoreductase polypeptide is capable ofconverting the substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine tothe product (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine, with apercent stereomeric excess of at least about 99% and at a rate that isat least 1500% improved over a reference polypeptide having the aminoacid sequence of SEQ ID NO:6. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides which compriseamino acid sequences corresponding to SEQ ID NO: 24, 26, 28, 30, and 32.

In some embodiments, the ketoreductase polypeptide is capable ofconverting at least about 95% of the monomethyl substrate to themonomethyl product in less than about 24 hours when carried out withgreater than about 100 g/L of substrate and less than about 5 g/L of thepolypeptide. Exemplary polypeptides that have this capability include,but are not limited to, polypeptides which comprises amino acidsequences corresponding to SEQ ID NO: 24, 26, 28, 30, and 32.

In some embodiments, the ketoreductase polypeptides of the disclosureare highly stereoselective and can reduce the monomethyl substrate tothe monomethyl product in greater than about 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% stereomeric excess. Exemplaryketoreductase polypeptides with such high stereoselectivity include, butare not limited to, the polypeptides comprising the amino acid sequencescorresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, and 32.

Table 2 below provides a list of the SEQ ID NOs disclosed herein withassociated activity levels with respect to the reduction of themonomethyl substrate to the corresponding monomethyl product. Thesequences below are also derived from the wild-type L. kefirketoreductase sequences (SEQ ID NO: 1 and 2) unless otherwise specified.In Table 2 below, each row lists two SEQ ID NOs, where the odd numberrefers to the nucleotide sequence that codes for the amino acid sequenceprovided by the even number. The column listing the number of mutationsis with respect to the number of amino acid substitutions as compared tothe L. kefir KRED amino acid sequence of SEQ ID NO:2. In the activitycolumn, one “+” indicates that the enzyme's ketoreductase activity is5-10 times greater than that of the enzyme of SEQ ID NO:6. Similarly, atwo plus sign symbol “++” indicates that the polypeptide ketoreductaseactivity is from 10 to about 15 times greater that the reference enzymeSEQ ID NO:6, and the three plus sign symbol “+++” indicates that thepolypeptide ketoreductase activity is more than 15 times greater thanthe activity of the reference enzyme of SEQ ID NO: 6.

TABLE 2 List of Sequences and Corresponding Activity Improvement withrespect to reduction of N-methyl-3-keto-3-(2-thienyl)-1-propanamine(“monomethyl substrate”): SEQ ID NO Number of mutations (Polynucleo-Residue Changes relative to the Activity tide/amino Relative towild-type L. kefir Monomethyl acid) SEQ ID NO: 2 sequence (SEQ ID NO: 2)Substrate 5/6 H40R; A94G; S96V; 12 “Control” E145F; F147M; Y190P; L195M;V196L; M206W; I226V; D233G; Y249W 7/8 H40R; A94D; S96V; 12 + E145F;F147M; Y190P; L195M; V196L; M206W; I226V; D233G; Y249W  9/10 H40R; A94D;S96V; 13 + K109E; E145F; F147M Y190P; L195M; V196L; M206W; I226V; D233G;Y249W 11/12 H40R; K46R; A94D; 16 ++ S96V; E145F; F147M; L153V; Y190P;L195M; V196L; L199V; M206W; I226V; D233G; V245I; Y249W 13/14 H40R; K46R;A94D; 15 ++ E145F; F147M; L153V; Y190P; L195M; V196L; L199V; M206W;I226V; D233G; V245I; Y249W 15/16 H40R; K46R; A94D; 15 ++ E145F; F147M;L153V; Y190P; L195M; V196L; L199R; M206W; I226V; D233G; V245I; Y249W17/18 H40R; K46R; A94D; S96P; 16 ++ E145F; F147M; L153V; Y190P; L195M;V196L; L199V; M206W; I226V; D233G; V245I; Y249W 19/20 H40R; K46R; A94D;16 + S96G; E145F; F147M; L153V; Y190P; L195M; V196L; L199V; M206W;I226V; D233G; V245I; Y249W 21/22 H40R; K46R; A94D; 16 ++ S96V; E145F;F147M; L153V; Y190P; L195M; V196L; L199E; M206W; I226V; D233G; V245I;Y249W 23/24 H40R; K46R; A94D; 17 +++ S96V; E145F; F147M; L153V; Y190P;L195M; V196L; D198N, L199V; M206W; I226V; D233G; V245I; Y249W 25/26H40R; K46R; A94D; 17 +++ S96V; E145F; F147M; L153V; N157S, Y190P; L195M;V196L; L199V; M206W; I226V; D233G; V245I; Y249W 27/28 H40R; K46R; V60I,A94D; 18 +++ S96V; R108H, E145F; F147M; L153V; Y190P; L195M; V196L;L199V; M206W; I226V; D233G; V245I; Y249W 29/30 H40R; K46R; A64V, 17 +++A94D; S96V; E145F; F147M; L153V; Y190P; L195M; V196L; L199V; M206W;I226V; D233G; V245I; Y249W 31/32 H40R; K46R; A94D; 17 +++ S96V; E145F;F147M; T152N, L153V; Y190P; L195M; V196L; L199E; M206W; I226V; D233G;V245I; Y249W

In some embodiments, the improved ketoreductase polypeptide hereincomprises an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalas compared a reference sequence based on SEQ ID NO:2, 4 or 60 havingthe following features described herein, for example: the amino acidresidue corresponding to position 94 is an acidic residue; the aminoacid residue corresponding to position 145 is aromatic residue orleucine; and the amino acid corresponding to position 190 is cysteine orproline, and wherein the ketoreductase polypeptides have at least thepreceding features. In some embodiments, the improved ketoreductaseshave the specified sequence identity based on a reference ketoreductaseand have the following features: the amino acid residue corresponding toposition 94 is an aspartic acid; the amino acid residue corresponding toposition 145 is phenylalanine, tyrosine, tryptophan, or leucine,particularly phenylalanine or leucine; and the amino acid correspondingto position 190 is cysteine or proline, particularly proline, andwherein the ketoreductase polypeptides have at least the precedingfeatures. In some embodiments, these ketoreductase polypeptides can haveone or more modifications as compared to the reference amino acidsequence. The modifications can include substitutions, deletions, andinsertions. The substitutions can be non-conservative substitutions,conservative substitutions, or a combination of non-conservative andconservative substitutions. In some embodiments, these ketoreductasepolypeptides can have optionally from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-25, 1-30, or about 1-35 mutations at other amino acid residues.In some embodiments, the number of mutations can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40mutations at other amino acid residues.

In some embodiments, an improved ketoreductase polypeptide comprises anamino acid sequence that corresponds to the sequence formula aspresented in SEQ ID NO:61, 62, or 63, or a region thereof, such asresidues 90-211. SEQ ID NO:61 is based on the amino acid sequence of theLactobacillus kefir ketoreductase (SEQ ID NO:2); SEQ ID NO:62 is basedon the amino acid sequence of the Lactobacillus brevis ketoreductase(SEQ ID NO:4); and SEQ ID NO:63 is based on the amino acid sequence ofthe Lactobacillus minor ketoreductase (SEQ ID NO:60). SEQ ID NOs:61, 62and 63 specify that residue corresponding to X94 is an acidic aminoacid, residue corresponding to X145 is an aromatic amino acid orleucine, and residue corresponding to X190 is cysteine or a constrainedamino acid. In some embodiments, the ketoreductase polypeptide based onthe sequence formula of SEQ ID NOs:61, 62 or 63 can comprise an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference aminoacid sequence based on SEQ ID NO:2, 4, or 60 having at least thefeatures described herein for amino acid residues X94, X145, and X190,with the proviso that the ketoreductase polypeptides have at least thespecified features. In some embodiments, the amino acid sequence of theketoreductase polypeptides have at least the following features: theamino acid residue corresponding to X94 is aspartic acid; the amino acidresidue corresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine or leucine; and the amino acid correspondingto X190 is cysteine or proline, particularly proline, with the provisothat the ketoreductase polypeptides have at least the specifiedfeatures.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62or 63, or region thereof, such as residue 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, canfurther include one or more features selected from the following:residue corresponding to X7 is nonpolar or constrained residue; residuecorresponding to X40 is a constrained, hydrophilic or basic residue;residue corresponding to X46 is a hydrophilic or basic residue; residuecorresponding to X60 is an aliphatic or non polar residue; residuecorresponding to X64 is an aliphatic or non-polar residue; residuecorresponding to X96 is a polar, constrained, non-polar or aliphaticresidue; residue corresponding to X108 is a hydrophilic, polar orconstrained residue; residue corresponding to X109 is an acidic residue;residue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; residue corresponding to X152 is a polar residue;residue corresponding to X153 is a polar, non-polar, or aliphaticresidue; residue corresponding to X157 is a polar residue; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue;residue corresponding to X196 is a non polar or aliphatic residue;residue corresponding to X198 is an acidic, polar residue, hydrophilicresidue; residue corresponding to X199 is an acidic, basic, hydrophilic,aliphatic or nonpolar residue; residue corresponding to X206 is anonpolar, aromatic, or hydrophobic residue; residue corresponding toX226 is a non polar or aliphatic residue; residue corresponding to X233is an acidic, non-polar, or aliphatic residue; residue corresponding toX245 is a non polar or aliphatic residue; and residue corresponding toX249 is a nonpolar or aromatic residue. In some of the foregoingembodiments, the amino acid residue corresponding to X94 is asparticacid; the amino acid residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine; and the amino acidcorresponding to X190 is cysteine or proline, particularly proline. Insome embodiments, the polypeptides comprising an amino acid sequencethat corresponds to the sequence formula provided in SEQ ID NO:61, 62 or63, or region thereof, can have additionally one or more of the residuesnot specified by an X to be mutated as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the mutations canbe from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40mutations at other amino acid residues not defined by X above. In someembodiments, the number of mutations can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 aminoacid residues. In some embodiments, the mutations comprise conservativemutations.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62and 63, or region thereof, such as residue 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, can haveone or more conservative mutations as compared to the amino acidsequence of SEQ ID NO:2, 4, or 60. Examples of such conservativemutations include amino acid replacements such as, but not limited to,replacement of residue X46 lysine (K) with another hydrophilic or basicresidue, e.g. arginine (R); replacement of residue X60 valine (V) withanother aliphatic or non-polar residue, e.g. isoleucine (I); replacementof residue X64 alanine (A) with another aliphatic or non-polar residue,e.g. valine (V); replacement of residue X152 threonine (T) with anotherpolar residue, e.g., asparagine (N); replacement of residue X153 withanother aliphatic or non-polar residue, e.g., valine; replacement ofresidue X157 asparagine (N) with another polar residue, e.g., serine(S); replacement of residue 185 threonine (T) with another polarresidue, e.g., serine (S); replacement of residue X195 leucine (L) withanother aliphatic or non-polar residue, e.g., methionine (M);replacement of residue X196 valine (V) with another non polar,hydrophobic, or aliphatic residue, e.g., leucine (L) or isoleucine (I);replacement of residue X199 leucine (L) with another non-polar oraliphatic residue, e.g., valine (V); replacement of residue 226isoleucine (I) with another non-polar or aliphatic residue, e.g. valine(V); replacement of residue 245 valine (V) with another non-polar oraliphatic residue, e.g. isoleucine (I); and replacement of residue 249tyrosine (Y) with another hydrophobic or aromatic residue, e.g.tryptophan (W).

In some embodiments, the improved ketoreductase polypeptides based onthe sequence formula of SEQ ID NO: 61, 62 or 63, or a region thereof,such as residues 90-211, having the features at residues X94, X145, andX190 as described herein, can further include one or more the featuresselected from the following: residue corresponding to X7 is glycine,methionine, alanine, valine, leucine, isoleucine, proline or histidine,particularly histidine; residue corresponding to X40 is threonine,serine, histidine, glutamic acid, asparagine, glutamine, aspartic acid,lysine, or arginine, particularly arginine; residue corresponding to X46is arginine or lysine; residue corresponding to X60 is glycine,methionine, alanine, valine, leucine, or isoleucine, particularlyisoleucine; residue corresponding to X64 is glycine, methionine,alanine, valine, leucine, isoleucine, particularly valine; residuecorresponding to X96 is proline, serine, threonine, asparagine,glutamine, glycine, methionine, alanine, valine, leucine, or isoleucine,particularly valine, proline, or glycine; residue corresponding to X108is threonine, serine, histidine, glutamic acid, asparagine, glutamine,aspartic acid, lysine, particularly histidine; residue corresponding toX109 is aspartic or glutamic acid; residue corresponding to X147 isisoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine and tyrosine, particularly isoleucine, methionine, valine, orleucine; residue corresponding to X152 is serine, threonine, asparagineor glutamine, particularly asparagine; residue corresponding to X153 isalanine, valine, leucine, isoleucine, serine, threonine, asparagine, orglutamine, particularly valine; residue corresponding to X157 is serine,threonine, asparagine, or glutamine, particularly serine; residuecorresponding to X195 is glycine, methionine, alanine, valine, leucine,or isoleucine, particularly methionine; residue corresponding to X196 isglycine, methionine, alanine, valine, leucine, isoleucine, particularlyleucine; residue corresponding to X198 is aspartic acid, glutamic acid,serine, threonine, asparagine, or glutamine, particularly asparagine;residue corresponding to X199 is aspartic acid, glutamic acid, arginine,lysine, serine, threonine, asparagine, glutamine, glycine, methionine,alanine, valine, leucine, or isoleucine, particularly valine, arginine,or glutamic acid; residue corresponding to X206 is isoleucine,phenylalanine, valine, leucine, tryptophan, methionine, alanine ortyrosine, particularly tyrosine, tryptophan, or phenylalanine; residuecorresponding to X226 is glycine, methionine, alanine, valine, leucine,or isoleucine, particularly valine; residue corresponding to X233 isglycine, methionine, alanine, valine, leucine, isoleucine, asparticacid, or glutamic acid, particularly glycine; residue corresponding toX245 is glycine, methionine, alanine, valine, leucine, or isoleucine,particularly isoleucine; and residue corresponding to X249 isisoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine or tyrosine, particularly tryptophan. In some embodiments, thepolypeptides comprising an amino acid sequence that corresponds to thesequence formula provided in SEQ ID NO: 61, 62 or 63 (or region thereof)can have additionally one or more of the residues not specified by an Xto be mutated as compared to the reference sequence of SEQ ID NO: 2, 4or 60. In some embodiments, the mutations can be 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 mutations at other amino acidresidues not defined by X above. In some embodiments, the number ofmutations can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18,20, 22, 24, 26, 30, 35 or about 40 mutations at other amino acidresidues. In some embodiments, the mutations comprise conservativemutations.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62or 63, or region thereof, such as residues 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, canfurther include one or more or at least all of the following features:residue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; residue corresponding to X206 is nonpolar,aromatic, or hydrophobic residue; and residue corresponding to X233 isan acidic, non-polar, or aliphatic residue. In some of the foregoingembodiments, the amino acid residue corresponding to X94 is asparticacid; the amino acid residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; and theamino acid corresponding to X190 is cysteine or proline, particularlyproline. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62or 63, or region thereof, such as residues 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, canfurther include one or more or at least all of the following features:residue corresponding to X40 is a constrained, hydrophilic or basicresidue; residue corresponding to X96 is a polar, constrained, non-polaror aliphatic residue; residue corresponding to X147 is an aromatic,non-polar, aliphatic, or hydrophobic residue; residue corresponding toX199 is an acidic, basic, hydrophilic, aliphatic or nonpolar residue;residue corresponding to X206 is a nonpolar, aromatic, or hydrophobicresidue; and residue corresponding to X233 is an acidic, non-polar, oraliphatic residue. In some of the foregoing embodiments, the amino acidresidue corresponding to X94 is aspartic acid; the amino acid residuecorresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine or leucine; and the amino acid correspondingto X190 is cysteine or proline, particularly proline. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62or 63, or region thereof, such as residues 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, canfurther include one or more or at least all of the following features:residue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; residue corresponding to X195 is a non-polar,aliphatic, or basic residue; residue corresponding to X196 is a nonpolar or aliphatic residue; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue; residue corresponding to X226 is a nonpolar or aliphatic residue; residue corresponding to X233 is an acidic,non-polar, or aliphatic residue; and residue corresponding to X249 is anonpolar or aromatic residue. In some of the foregoing embodiments, theamino acid residue corresponding to X94 is aspartic acid; the amino acidresidue corresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine or leucine; and the amino acid correspondingto X190 is cysteine or proline, particularly proline. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, the improved ketoreductase polypeptides based onthe sequence formula of SEQ ID NO: 61, 62 or 63, or a region thereof,such as residues 90-211, having the features described herein atresidues X94, X145, and X190 as described herein, can further includeone or more or at least all of the following features: residuecorresponding to X147 is isoleucine, phenylalanine, valine, leucine,tryptophan, methionine, alanine and tyrosine, particularly isoleucine,methionine, valine, or leucine; residue corresponding to X195 isglycine, methionine, alanine, valine, leucine, or isoleucine,particularly methionine; residue corresponding to X196 is glycine,methionine, alanine, valine, leucine, isoleucine, particularly leucine;residue corresponding to X206 is isoleucine, phenylalanine, valine,leucine, tryptophan, methionine, alanine or tyrosine, particularlytyrosine, tryptophan, or phenylalanine; residue corresponding to X226 isglycine, methionine, alanine, valine, leucine, or isoleucine,particularly valine; residue corresponding to X233 is glycine,methionine, alanine, valine, leucine, isoleucine, aspartic acid, orglutamic acid, particularly glycine; and residue corresponding to X249is isoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine or tyrosine, particularly tryptophan. In some of the foregoingembodiments, and the amino acid residue corresponding to X94 is asparticacid; the amino acid residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; and theamino acid corresponding to X190 is cysteine or proline, particularlyproline. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, the improved ketoreductase polypeptides comprisingan amino acid sequence based on the sequence formula of SEQ ID NO:61, 62or 63, or region thereof, such as residue 90-211, having the specifiedfeatures for residues X94, X145, and X190 as described herein, canfurther include at one or more or at least all of the followingfeatures: residue corresponding to X40 is a constrained, hydrophilic orbasic residue; residue corresponding to X46 is a hydrophilic or basicresidue; residue corresponding to X96 is a polar, constrained, non-polaror aliphatic residue; residue corresponding to X147 is an aromatic,non-polar, aliphatic, or hydrophobic residue; residue corresponding toX153 is a polar, non-polar, or aliphatic residue; residue correspondingto X195 is a non-polar, aliphatic, or basic residue; residuecorresponding to X196 is a non polar or aliphatic residue; residuecorresponding to X199 is an acidic, basic, hydrophilic, aliphatic ornonpolar residue; residue corresponding to X206 is a nonpolar, aromatic,or hydrophobic residue; residue corresponding to X226 is a non polar oraliphatic residue; residue corresponding to X233 is an acidic,non-polar, or aliphatic residue; residue corresponding to X245 is a nonpolar or aliphatic residue; and residue corresponding to X249 is anonpolar or aromatic residue. In some of the foregoing embodiments, theamino acid residue corresponding to X94 is aspartic acid; the amino acidresidue corresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine or leucine; and the amino acid correspondingto X190 is cysteine or proline, particularly proline. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, the improved ketoreductase polypeptides based onthe sequence formula of SEQ ID NO: 61, 62 or 63, or a region thereof,such as residues 90-211, having the features described herein atresidues X94, X145, and X190 as described herein, can further includeone or more or at least all of the following features: residuecorresponding to X40 is threonine, serine, histidine, glutamic acid,asparagine, glutamine, aspartic acid, lysine, or arginine, particularlyarginine; residue corresponding to X46 is arginine or lysine; residuecorresponding to X96 s proline, serine, threonine, asparagine,glutamine, glycine, methionine, alanine, valine, leucine, or isoleucine,particularly valine, proline, or glycine; residue corresponding to X147s isoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine and tyrosine, particularly isoleucine, methionine, valine, orleucine; residue corresponding to X153 is alanine, valine, leucine,isoleucine, serine, threonine, asparagine, or glutamine, particularlyvaline; residue corresponding to X195 is glycine, methionine, alanine,valine, leucine, or isoleucine, particularly methionine; residuecorresponding to X196 is glycine, methionine, alanine, valine, leucine,isoleucine, particularly leucine; residue corresponding to X199 isaspartic acid, glutamic acid, arginine, lysine, serine, threonine,asparagine, glutamine, glycine, methionine, alanine, valine, leucine, orisoleucine, particularly valine, arginine, or glutamic acid; residuecorresponding to X206 is isoleucine, phenylalanine, valine, leucine,tryptophan, methionine, alanine or tyrosine, particularly tyrosine,tryptophan, or phenylalanine; residue corresponding to X226 is glycine,methionine, alanine, valine, leucine, or isoleucine, particularlyvaline; residue corresponding to X233 is glycine, methionine, alanine,valine, leucine, isoleucine, aspartic acid, or glutamic acid,particularly glycine; residue corresponding to X245 is glycine,methionine, alanine, valine, leucine, or isoleucine, particularlyisoleucine; and residue corresponding to X249 is isoleucine,phenylalanine, valine, leucine, tryptophan, methionine, alanine ortyrosine, particularly tryptophan. In some of the foregoing embodiments,the amino acid residue corresponding to X94 is aspartic acid; the aminoacid residue corresponding to X145 is phenylalanine, tyrosine, orleucine, particularly phenylalanine or leucine; and the amino acidcorresponding to X190 is cysteine or proline, particularly proline. Insome embodiments, the ketoreductase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X7 is a nonpolar or constrained residue,particularly a histidine. In some embodiments, the ketoreductasepolypeptides can have additionally from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35 or about 1-40 mutations at other amino acidresidues. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X40 is a constrained, hydrophilic or basicresidue, particularly arginine. In some embodiments, the ketoreductasepolypeptides can have additionally from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35 or about 1-40 mutations at other amino acidresidues. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X46 is a hydrophilic or basic residue,particularly arginine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X60 is an aliphatic or non polar residue,particularly isoleucine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X64 is an aliphatic or non-polar residue,particularly valine. In some embodiments, the ketoreductase polypeptidescan have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35or about 1-40 residue differences at other amino acid residues ascompared to the reference sequence of SEQ ID NO:2, 4 or 60. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with at leastthe preceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity as compared to a reference sequence based on SEQ ID NO:2, 4or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X96 is a polar, constrained, non-polar oraliphatic residue, particularly proline, glycine, and valine. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X108 is a hydrophilic, polar or constrainedresidue, particularly histidine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X109 is an acidic residue, particularlyglutamic acid. In some embodiments, the ketoreductase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 orabout 1-40 residue differences at other amino acid residues as comparedto the reference sequence of SEQ ID NO:2, 4 or 60. In some embodiments,the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differencesat other amino acid residues. In some embodiments, the differencescomprise conservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue, particularly methionine. In some embodiments, theketoreductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residue differences at otheramino acid residues as compared to the reference sequence of SEQ IDNO:2, 4 or 60. In some embodiments, the number of differences can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30,35 or about 40 residue differences at other amino acid residues. In someembodiments, the differences comprise conservative mutations. In someembodiments, the ketoreductase polypeptide comprises an amino acidsequence with at least the preceding features, and wherein the aminoacid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity as compared to a referencesequence based on SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X152 is a polar residue, particularlyasparagine. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X153 is a polar, non-polar, or aliphaticresidue, particularly valine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X157 is a polar residue, particularly serine orthreonine. In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40residue differences at other amino acid residues as compared to thereference sequence of SEQ ID NO:2, 4 or 60. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residue differences atother amino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X195 is a non-polar, aliphatic, or basicresidue, particularly methionine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X196 is a non polar or aliphatic residue,particularly leucine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X198 is an acidic, polar residue, hydrophilicresidue, particularly asparagine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X199 is acidic, basic, hydrophilic, aliphaticor nonpolar residue, particularly valine, glutamic acid, or arginine. Insome embodiments, the ketoreductase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X206 is a nonpolar, aromatic, or hydrophobicresidue, particularly phenylalanine, tyrosine, or tryptophan. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35 or about 1-40 residuedifferences at other amino acid residues as compared to the referencesequence of SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 residue differences at otheramino acid residues. In some embodiments, the differences compriseconservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to a reference sequence based on SEQ ID NO:2, 4 or60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X226 is a non polar or aliphatic residue,particularly valine. In some embodiments, the ketoreductase polypeptidescan have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35or about 1-40 residue differences at other amino acid residues ascompared to the reference sequence of SEQ ID NO:2, 4 or 60. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with at leastthe preceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity as compared to a reference sequence based on SEQ ID NO:2, 4or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X233 is an acidic, non-polar, or aliphaticresidue, particularly glycine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: residue corresponding toX94 is aspartic acid; residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine or leucine; residuecorresponding to X190 is cysteine or proline, particularly proline; andresidue corresponding to X245 is a non polar or aliphatic residue,particularly isoleucine. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence based on the sequence formula of SEQ ID NO: 61, 62 or 63, or aregion thereof, such as residues 90-211, in which the amino acidsequence has at least the following features: amino acid residuecorresponding to X94 is aspartic acid; the amino acid residuecorresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine or leucine; the amino acid corresponding toX190 is cysteine or proline, particularly proline; and the amino acidresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35 or about 1-40 residue differences at other amino acidresidues as compared to the reference sequence of SEQ ID NO:2, 4 or 60.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with atleast the preceding features, and wherein the amino acid sequence has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity as compared to a reference sequence based on SEQ IDNO:2, 4 or 60 with the preceding features.

In some embodiments, an improved ketoreductase comprises an amino acidsequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence corresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, and 32, as listed in Table 2, wherein the improvedketoreductase polypeptide amino acid sequence includes any one set ofthe specified amino acid substitution combinations presented in Table 2.In some embodiments, the ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40differences at other amino acid residues as compared to the referencesequence. In some embodiments, the number of differences can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 orabout 40 residue differences at other amino acid residues. In someembodiments, the differences comprise conservative mutations.

In some embodiments, the ketoreductases of the disclosure are subject tothe proviso that they do not include the specific sequencescorresponding to SEQ ID NO:12 and SEQ ID NO:22. Thus, each and every ofthe embodiments described herein has the proviso that it excludes thespecific polypeptides defined by SEQ ID NO: 12 and 22.

In some embodiments, the improved ketoreductases comprise amino acidsequences corresponding to SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, and 32.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, and has at leastthe following features: residue corresponding to X40 is a constrained,hydrophilic or basic residue, particularly arginine; residuecorresponding to X94 is an acidic residue, particularly aspartic acid;residue corresponding to X96 is a polar, constrained, non-polar oraliphatic residue; residue corresponding to X145 is an aromatic residueor leucine; residue corresponding to X147 is an aromatic, non-polar,aliphatic, or hydrophobic residue, particularly methionine; residuecorresponding to X190 is cysteine or a constrained residue, particularlyproline; residue corresponding to X195 is a non-polar, aliphatic, orbasic residue, particularly methionine; residue corresponding to X196 isa non polar or aliphatic residue, particularly leucine; residuecorresponding to X206 is a nonpolar, aromatic, or hydrophobic residue,particularly tryptophan; residue corresponding to X226 is a non polar oraliphatic residue, particularly valine; residue corresponding to X233 isan acidic, non-polar, or aliphatic residue, particularly glycine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:10. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:10.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyvaline; residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or a constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X199is an acidic, basic, hydrophilic, aliphatic or nonpolar residue,particularly valine; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue, particularly tryptophan; residuecorresponding to X226 is a non polar or aliphatic residue, particularlyvaline; residue corresponding to X233 is an acidic, non-polar, oraliphatic residue, particularly glycine; residue corresponding to X245is a non polar or aliphatic residue, particularly isoleucine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:12. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:12.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding toX145 is an aromatic residue or leucine, particularly phenylalanine;residue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue, particularly methionine; residue corresponding toX153 is a polar, non-polar, or aliphatic residue, particularly valine;residue corresponding to X190 is cysteine or a constrained residue,particularly proline; residue corresponding to X195 is a non-polar,aliphatic, or basic residue, particularly methionine; residuecorresponding to X196 is a non polar or aliphatic residue, particularlyleucine; residue corresponding to X199 is an acidic, basic, hydrophilic,aliphatic or nonpolar residue, particularly valine or arginine; residuecorresponding to X206 is a nonpolar, aromatic, or hydrophobic residue,particularly tryptophan; residue corresponding to X226 is a non polar oraliphatic residue, particularly valine; residue corresponding to X233 isan acidic, non-polar, or aliphatic residue, particularly glycine;residue corresponding to X245 is a non polar or aliphatic residue,particularly isoleucine, and residue corresponding to X249 is a nonpolaror aromatic residue, particularly tryptophan. In some embodiments, theketoreductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 residue differences at otherresidue positions as compared to a reference sequence of SEQ ID NO:14 or16. In some embodiments, the number of differences can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about40 residue differences at other amino acid residues. In someembodiments, the differences comprise conservative mutations. In someembodiments, the ketoreductase polypeptide comprises an amino acidsequence with the preceding features, and wherein the amino acidsequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:14 or 16.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyproline or glycine; residue corresponding to X145 is an aromatic residueor leucine, particularly phenylalanine; residue corresponding to X147 isan aromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X199is an acidic, basic, hydrophilic, aliphatic or nonpolar residue,particularly valine; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue, particularly tryptophan; residuecorresponding to X226 is a non polar or aliphatic residue, particularlyvaline; residue corresponding to X233 is an acidic, non-polar, oraliphatic residue, particularly glycine; residue corresponding to X245is a non polar or aliphatic residue, particularly isoleucine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:18 or 20. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:18 or 20.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyvaline; residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X199 is an acidic,basic, hydrophilic, aliphatic or nonpolar residue, particularly glutamicacid; residue corresponding to X206 is a nonpolar, aromatic, orhydrophobic residue, particularly tryptophan; residue corresponding toX226 is a non polar or aliphatic residue, particularly valine; residuecorresponding to X233 is an acidic, non-polar, or aliphatic residue,particularly glycine; residue corresponding to X245 is a non polar oraliphatic residue, particularly isoleucine; and residue corresponding toX249 is a nonpolar or aromatic residue, particularly tryptophan. In someembodiments, the ketoreductase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 residuedifferences at other residue positions as compared to a referencesequence of SEQ ID NO:22. In some embodiments, the number of differencescan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22,24, 26, 30, 35 or about 40 residue differences at other amino acidresidues. In some embodiments, the differences comprise conservativemutations. In some embodiments, the ketoreductase polypeptide comprisesan amino acid sequence with the preceding features, and wherein theamino acid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:22.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyvaline; residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X198is an acidic, polar residue, hydrophilic residue, particularlyasparagine; residue corresponding to X199 is an acidic, basic,hydrophilic, aliphatic or nonpolar residue, particularly valine; residuecorresponding to X206 is a nonpolar, aromatic, or hydrophobic residue,particularly tryptophan; residue corresponding to X226 is a non polar oraliphatic residue, particularly valine; residue corresponding to X233 isan acidic, non-polar, or aliphatic residue, particularly glycine;residue corresponding to X245 is a non polar or aliphatic residue,particularly isoleucine; and residue corresponding to X249 is a nonpolaror aromatic residue, particularly tryptophan. In some embodiments, theketoreductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 residue differences at otherresidue positions as compared to a reference sequence of SEQ ID NO:22.In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40residue differences at other amino acid residues. In some embodiments,the differences comprise conservative mutations. In some embodiments,the ketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:24.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyvaline; residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X157 isa polar residue, particularly serine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X199is an acidic, basic, hydrophilic, aliphatic or nonpolar residue,particularly valine; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue, particularly tryptophan; residuecorresponding to X226 is a non polar or aliphatic residue, particularlyvaline; residue corresponding to X233 is an acidic, non-polar, oraliphatic residue, particularly glycine; residue corresponding to X245is a non polar or aliphatic residue, particularly isoleucine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:26. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:26.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X60 is analiphatic or non polar residue, particularly isoleucine; residuecorresponding to X94 is an acidic residue, particularly aspartic acid;residue corresponding to X96 is a polar, constrained, non-polar oraliphatic residue, particularly valine; residue corresponding to X108 isa hydrophilic, polar or constrained residue, particularly histidine;residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X199is an acidic, basic, hydrophilic, aliphatic or nonpolar residue,particularly valine; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue, particularly tryptophan; residuecorresponding to X226 is a non polar or aliphatic residue, particularlyvaline; residue corresponding to X233 is an acidic, non-polar, oraliphatic residue, particularly glycine; residue corresponding to X245is a non polar or aliphatic residue, particularly isoleucine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:28. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:28.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X64 is analiphatic or non-polar residue, particularly valine; residuecorresponding to X94 is an acidic residue, particularly aspartic acid;residue corresponding to X96 is a polar, constrained, non-polar oraliphatic residue, particularly valine; residue corresponding to X108 ishydrophilic, polar or constrained residue, particularly histidine;residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X153 is a polar, non-polar, oraliphatic residue, particularly valine; residue corresponding to X190 iscysteine or constrained residue, particularly proline; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue,particularly methionine; residue corresponding to X196 is a non polar oraliphatic residue, particularly leucine; residue corresponding to X199is an acidic, basic, hydrophilic, aliphatic or nonpolar residue,particularly valine; residue corresponding to X206 is a nonpolar,aromatic, or hydrophobic residue, particularly tryptophan; residuecorresponding to X226 is a non polar or aliphatic residue, particularlyvaline; residue corresponding to X233 is an acidic, non-polar, oraliphatic residue, particularly glycine; residue corresponding to X245is a non polar or aliphatic residue, particularly isoleucine; andresidue corresponding to X249 is a nonpolar or aromatic residue,particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:30. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:30.

In some embodiments, an improved ketoreductase polypeptides comprises anamino acid sequence based on the sequence formula of SEQ ID NO: 61, 62or 63, or a region thereof, such as residues 90-211, in which the aminoacid sequence has at least the following features: residue correspondingto X40 is a constrained, hydrophilic or basic residue, particularlyarginine; residue corresponding to X46 is a hydrophilic or basicresidue, particularly arginine; residue corresponding to X94 is anacidic residue, particularly aspartic acid; residue corresponding to X96is a polar, constrained, non-polar or aliphatic residue, particularlyvaline; residue corresponding to X145 is an aromatic residue or leucine,particularly phenylalanine; residue corresponding to X147 is anaromatic, non-polar, aliphatic, or hydrophobic residue, particularlymethionine; residue corresponding to X152 is a polar residue,particularly asparagine; residue corresponding to X153 is a polar,non-polar, or aliphatic residue, particularly valine; residuecorresponding to X190 is cysteine or constrained residue, particularlyproline; residue corresponding to X195 is a non-polar, aliphatic, orbasic residue, particularly methionine; residue corresponding to X196 isa non polar or aliphatic residue, particularly leucine; residuecorresponding to X199 is an acidic, basic, hydrophilic, aliphatic ornonpolar residue, particularly valine; residue corresponding to X206 isa nonpolar, aromatic, or hydrophobic residue, particularly tryptophan;residue corresponding to X226 is a non polar or aliphatic residue,particularly valine; residue corresponding to X233 is an acidic,non-polar, or aliphatic residue, particularly glycine; residuecorresponding to X245 is a non polar or aliphatic residue, particularlyisoleucine; and residue corresponding to X249 is a nonpolar or aromaticresidue, particularly tryptophan. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25,1-30, 1-35 or about 1-40 residue differences at other residue positionsas compared to a reference sequence of SEQ ID NO:32. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 residuedifferences at other amino acid residues. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with thepreceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO:32.

In some embodiments, an improved ketoreductase comprises an amino acidsequence that has a region or domain corresponding to residues 90-211 ofsequence formula of SEQ ID NO: 61, 62 or 63, in which the amino acidsequence of the domain has at least the following features: (1) theamino acid residue corresponding to residue X94 is an acidic residue,(2) the amino acid residue corresponding to residue X145 is an aromaticresidue or leucine, and (3) the amino acid residue corresponding toresidue X190 is a cysteine or a constrained residue. In someembodiments, the region or domain that corresponds to residues 90-211 ofsequence formula of SEQ ID NO: 61, 62 or 63 has at least the followingfeatures in the domain: (1) the amino acid residue corresponding to X94is aspartic acid or glutamic acid, (2) the amino acid residuecorresponding to X145 is tyrosine, tryptophan, phenylalanine, orleucine, and (3) the amino acid residue corresponding to X190 iscysteine or proline. In some embodiments, the region or domain thatcorresponds to residues 90-211 of sequence formula of SEQ ID NO: 61, 62or 63 has at least the following features: (1) the amino acid residuecorresponding to position 94 is aspartic acid, (2) the amino acidresidue corresponding to position 145 is phenylalanine, tyrosine, orleucine, particularly phenylalanine or leucine, and (3) the amino acidcorresponding to position 190 is proline. In some embodiments, theregion or domain corresponding to residues 90-211 can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, or 1-20 residue differences at other amino acid residues ascompared to the corresponding domain of a reference sequence based onSEQ ID NO: 2, 4, or 60. In some embodiments, the number of differencescan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or about20 residue differences at other amino acid residues in the domain. Insome embodiments, the differences comprise conservative mutations. Insome embodiments, the ketoreductase polypeptide comprises an amino acidsequence with at least the preceding features, and wherein the aminoacid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity as compared to the amino acidsequence corresponding to residues 90-211 of a reference sequence basedon SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe domain one or more features selected from the following: residuecorresponding to X96 is a polar, constrained, non-polar or aliphaticresidue; residue corresponding to X108 is hydrophilic, polar orconstrained residue; residue corresponding to X109 is an acidic residue;residue corresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; residue corresponding to X152 is a polar residue;residue corresponding to X153 is a polar, non-polar, or aliphaticresidue; residue corresponding to X157 is a polar residue; residuecorresponding to X195 is a non-polar, aliphatic, or basic residue;residue corresponding to X196 is a non polar or aliphatic residue;residue corresponding to X198 is an acidic, polar residue, hydrophilicresidue; residue corresponding to X199 is an acidic, basic, hydrophilic,aliphatic or nonpolar residue; and residue corresponding to X206 is anonpolar, aromatic, or hydrophobic residue. In some of the foregoingembodiments, the residue corresponding to X94 is aspartic acid; theresidue corresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine; and the residue corresponding to X190 iscysteine or proline, particularly proline. In some embodiments, thedomain or region corresponding to residues 90-211 can have additionallyone or more of the residues not specified by an X to be conservativelymutated. In some embodiments, the region or domain corresponding toresidues 90-211 can have additionally from about 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, or 1-20residue differences at other amino acid residues as compared to thecorresponding domain of a reference sequence based on SEQ ID NO:2, 4 or60. In some embodiments, the number of differences can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or about 20 residue differencesat other amino acid residues in the domain. In some embodiments, thedifferences comprise conservative mutations.

In some embodiments, the ketoreductases polypeptides having a domainwith an amino acid sequence corresponding to residues 90-211 based onsequence formula of SEQ ID NO: 61, 62, or 63, as described above, wherethe domain can have one or more conservative mutations as compared tothe amino acid sequences of the corresponding domain of SEQ ID NO: 2, 4or 60. Examples of such conservative mutations include amino acidreplacements such as, but limited to: replacement of residue X152threonine (T) with another polar residue, e.g., asparagine (N);replacement of residue X153 with another aliphatic or non-polar residue,e.g., valine; replacement of residue X157 asparagine (N) with anotherpolar residue, e.g., serine (S); replacement of residue 185 threonine(T) with another polar residue, e.g., serine (S); replacement of residueX195 leucine (L) with another aliphatic or non-polar residue, e.g.,methionine (M); replacement of residue X196 valine (V) with another nonpolar, hydrophobic, or aliphatic residue, e.g., leucine (L) orisoleucine (I); replacement of residue X199 leucine (L) with anothernon-polar or aliphatic residue, e.g., valine (V).

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain one or more features selected from the following:residue corresponding to X96 is proline, serine, threonine, asparagine,glutamine, glycine, methionine, alanine, valine, leucine, or isoleucine,particularly valine, proline, or glycine; residue corresponding to X108is threonine, serine, histidine, glutamic acid, asparagine, glutamine,aspartic acid, lysine, particularly histidine; residue corresponding toX109 is aspartic or glutamic acid; residue corresponding to X147 isisoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine and tyrosine, particularly isoleucine, methionine, valine, orleucine; residue corresponding to X152 is serine, threonine, asparagineor glutamine, particularly asparagine; residue corresponding to X153 isalanine, valine, leucine, isoleucine, serine, threonine, asparagine, orglutamine, particularly valine; residue corresponding to X157 is serine,threonine, asparagine, or glutamine, particularly serine; residuecorresponding to X195 is glycine, methionine, alanine, valine, leucine,or isoleucine, particularly methionine; residue corresponding to X196 isglycine, methionine, alanine, valine, leucine, isoleucine, particularlyleucine; residue corresponding to X198 is aspartic acid, glutamic acid,serine, threonine, asparagine, or glutamine, particularly asparagine;residue corresponding to X199 is aspartic acid, glutamic acid, arginine,lysine, serine, threonine, asparagine, glutamine, glycine, methionine,alanine, valine, leucine, or isoleucine, particularly valine, arginine,or glutamic acid; residue corresponding to X206 is isoleucine,phenylalanine, valine, leucine, tryptophan, methionine, alanine ortyrosine, particularly tyrosine, tryptophan, or phenylalanine. In someembodiments, the region or domain corresponding to residues 90-211 canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, or 1-20 residue differences at other aminoacid residues as compared to the domain of a reference sequence based onSEQ ID NO:2, 4 or 60. In some embodiments, the number of differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or about 20residue differences at other amino acid residues in the domain. In someembodiments, the differences comprise conservative mutations.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; and residue corresponding to X206 is nonpolar,aromatic, or hydrophobic residue. In some embodiments, the region ordomain corresponding to residues 90-211 can have additionally 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18,or 1-20 residue differences at other amino acid residues as compared tothe domain of a reference sequence based on SEQ ID NO:2, 4 or 60. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, or about 20 residue differences atother amino acid residues in the domain. In some embodiments, thedifferences comprise conservative mutations. In some embodiments, theketoreductase polypeptide comprises an amino acid sequence with at leastthe preceding features, and wherein the amino acid sequence has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity as compared to the amino acid sequence corresponding toresidues 90-211 of a reference sequence based on SEQ ID NO:2, 4 or 60with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X96 is a polar, constrained, non-polar or aliphaticresidue; residue corresponding to X147 is an aromatic, non-polar,aliphatic, or hydrophobic residue; residue corresponding to X199 is anacidic, basic, hydrophilic, aliphatic or nonpolar residue; and residuecorresponding to X206 is a nonpolar, aromatic, or hydrophobic residue.In some embodiments, the region or domain corresponding to residues90-211 can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9,1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, or 1-20 residue differences atother amino acid residues as compared to the domain of a referencesequence based on SEQ ID NO:2, 4 or 60. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, or about 20 residue differences at other amino acid residues in thedomain. In some embodiments, the differences comprise conservativemutations. In some embodiments, the ketoreductase polypeptide comprisesan amino acid sequence with at least the preceding features, and whereinthe amino acid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as compared to theamino acid sequence corresponding to residues 90-211 of a referencesequence based on SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X147 is an aromatic, non-polar, aliphatic, orhydrophobic residue; residue corresponding to X195 is a non-polar,aliphatic, or basic residue; residue corresponding to X196 is a nonpolar or aliphatic residue; and residue corresponding to X206 is anonpolar, aromatic, or hydrophobic residue. In some of the foregoingembodiments, the amino acid residue corresponding to X94 is asparticacid; the amino acid residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine; and the amino acidcorresponding to X190 is cysteine or proline, particularly proline. Insome of the foregoing embodiments, the amino acid residue correspondingto X94 is aspartic acid; the amino acid residue corresponding to X145 isphenylalanine, tyrosine, or leucine, particularly phenylalanine orleucine; and the amino acid corresponding to X190 is cysteine orproline, particularly proline. In some embodiments, the region or domaincorresponding to residues 90-211 can have additionally 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, or1-20 residue differences at other amino acid residues as compared to thedomain of a reference sequence based on SEQ ID NO:2, 4 or 60. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, or about 20 residue differences at otheramino acid residues in the domain. In some embodiments, the differencescomprise conservative mutations. In some embodiments, the ketoreductasepolypeptide comprises an amino acid sequence with at least the precedingfeatures, and wherein the amino acid sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity as compared to the amino acid sequence corresponding toresidues 90-211 of a reference sequence based on SEQ ID NO:2, 4 or 60with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X147 is isoleucine, phenylalanine, valine, leucine,tryptophan, methionine, alanine and tyrosine, particularly isoleucine,methionine, valine, or leucine; residue corresponding to X195 isglycine, methionine, alanine, valine, leucine, or isoleucine,particularly methionine; residue corresponding to X196 s glycine,methionine, alanine, valine, leucine, isoleucine, particularly leucine;and residue corresponding to X206 is isoleucine, phenylalanine, valine,leucine, tryptophan, methionine, alanine or tyrosine, particularlytyrosine, tryptophan, or phenylalanine. In some of the foregoingembodiments, the amino acid residue corresponding to X94 is asparticacid; the amino acid residue corresponding to X145 is phenylalanine,tyrosine, or leucine, particularly phenylalanine; and the amino acidcorresponding to X190 is cysteine or proline, particularly proline. Insome embodiments, the region or domain corresponding to residues 90-211can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, or 1-20 residue differences at otheramino acid residues as compared to the domain of a reference sequencebased on SEQ ID NO:2, 4 or 60. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, or about 20 residue differences at other amino acid residues in thedomain. In some embodiments, the differences comprise conservativemutations. In some embodiments, the ketoreductase polypeptide comprisesan amino acid sequence with at least the preceding features, and whereinthe amino acid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as compared to theamino acid sequence corresponding to residues 90-211 of a referencesequence based on SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X96 is a polar, constrained, non-polar or aliphaticresidue; residue corresponding to X147 is an aromatic, non-polar,aliphatic, or hydrophobic residue; residue corresponding to X153 is apolar, non-polar, or aliphatic residue; residue corresponding to X195 isa non-polar, aliphatic, or basic residue; residue corresponding to X196is a non polar or aliphatic residue; residue corresponding to X199 is anacidic, basic, hydrophilic, aliphatic or nonpolar residue; and residuecorresponding to X206 is a nonpolar, aromatic, or hydrophobic residue.In some of the foregoing embodiments, the amino acid residuecorresponding to X94 is aspartic acid; the amino acid residuecorresponding to X145 is phenylalanine, tyrosine, or leucine,particularly phenylalanine; and the amino acid corresponding to X190 iscysteine or proline, particularly proline. In some embodiments, theregion or domain corresponding to residues 90-211 can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, or 1-20 residue differences at other amino acid residues ascompared to the domain of a reference sequence based on SEQ ID NO:2, 4or 60. In some embodiments, the number of differences can be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or about 20 residuedifferences at other amino acid residues in the domain. In someembodiments, the differences comprise conservative mutations. In someembodiments, the ketoreductase polypeptide comprises an amino acidsequence with at least the preceding features, and wherein the aminoacid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity as compared to the amino acidsequence corresponding to residues 90-211 of a reference sequence basedon SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, the ketoreductase polypeptides with a regioncorresponding to residues 90-211 and having the specified features forresidues X94, X145 and X190 as described herein, can further include inthe region or domain at least the following features: residuecorresponding to X96 s proline, serine, threonine, asparagine,glutamine, glycine, methionine, alanine, valine, leucine, or isoleucine,particularly valine, proline, or glycine; residue corresponding to X147is isoleucine, phenylalanine, valine, leucine, tryptophan, methionine,alanine and tyrosine, particularly isoleucine, methionine, valine, orleucine; residue corresponding to X153 is alanine, valine, leucine,isoleucine, serine, threonine, asparagine, or glutamine, particularlyvaline; residue corresponding to X195 is glycine, methionine, alanine,valine, leucine, or isoleucine, particularly methionine; residuecorresponding to X196 is glycine, methionine, alanine, valine, leucine,isoleucine, particularly leucine; residue corresponding to X199 isaspartic acid, glutamic acid, arginine, lysine, serine, threonine,asparagine, glutamine, glycine, methionine, alanine, valine, leucine, orisoleucine, particularly valine, arginine, or glutamic acid; and residuecorresponding to X206 is isoleucine, phenylalanine, valine, leucine,tryptophan, methionine, alanine or tyrosine, particularly tyrosine,tryptophan, or phenylalanine. In some of the foregoing embodiments, theamino acid residue corresponding to X94 is aspartic acid; the amino acidresidue corresponding to X145 is phenylalanine, tyrosine, or leucine,particular phenylalanine or leucine; and the amino acid corresponding toX190 is cysteine or proline, particularly proline. In some embodiments,the region or domain corresponding to residues 90-211 can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, or 1-20 residue differences at other amino acidresidues as compared to the domain of a reference sequence based on SEQID NO:2, 4 or 60. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or about 20residue differences at other amino acid residues in the domain. In someembodiments, the differences comprise conservative mutations. In someembodiments, the ketoreductase polypeptide comprises an amino acidsequence with at least the preceding features, and wherein the aminoacid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity as compared to the amino acidsequence corresponding to residues 90-211 of a reference sequence basedon SEQ ID NO:2, 4 or 60 with the preceding features.

In some embodiments, the ketoreductase polypeptides of the invention cancomprise an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence based on SEQ ID NO:2, 4 or 60, or a region ordomain thereof, such as residues 90-211, with the proviso that theresidue corresponding to position 94 is aspartic acid; the residuecorresponding to position 145 is phenylalanine, tyrosine or leucine,particularly phenylalanine or leucine; the residue corresponding toposition 190 is cysteine or proline, particularly proline; and whereinthe polypeptide can additionally have one or more of the followingsubstitutions such that the polypeptide is further improved (e.g., withrespect to stereoselectivity, enzymatic activity, and/orthermostability) over the wild-type kefir ketoreductase or anotherengineered ketoreductase, such as SEQ ID NO:6: 7→H; 40→R; 46→R; 60→I;64→V; 96→P,G,V; 108→H; 109→E; 147→M; 152→N,S; 153→A,V; 157→S; 195→R, M;196→L; 198→N; 199→V,E,R; 206→F,W,Y; 226→V; 233→G,A; 245→I; and 249→W.

In some embodiments, the ketoreductase polypeptides of the invention cancomprise a region having an amino acid sequence that is at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a reference sequence based on SEQ ID NO:2, 4 or 60, ora region or domain thereof, such as residues 90-211, with the provisothat the residue corresponding to position 94 is aspartic acid; theresidue corresponding to position 145 is phenylalanine; the residuecorresponding to position 190 is proline; and wherein the polypeptidecan additionally have one or more of the following substitutions suchthat the polypeptide is further improved (with respect tostereoselectivity, enzymatic activity, and/or thermostability) over thewild-type kefir ketoreductase or another engineered ketoreductase (e.g.,SEQ ID NO:6): 7→H; 40→R; 46→R; 60→I; 64→V; 96→P,G,V; 108→H; 109→E;147→M; 152→N; 153→A,V; 157→S; 195→M; 196→L; 198→N; 199→V,E,R; 206→W;226→V; 233→G; 245→I; and 249→W.

In some embodiments, the ketoreductase polypeptides of the invention cancomprise an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence based on SEQ ID NO:2, 4 or 60, or a region ordomain thereof, such as residues 90-211, with the proviso that theresidue corresponding to position 94 is aspartic acid; the residuecorresponding to position 145 is phenylalanine, tyrosine or leucine,particularly phenylalanine or leucine; the residue corresponding toposition 190 is cysteine or proline; the residue corresponding toposition 40 is arginine, and wherein the polypeptide can additionallyhave one or more of the following substitutions such that thepolypeptide is further improved (e.g., with respect tostereoselectivity, enzymatic activity, and/or thermostability) over thewild-type kefir ketoreductase or another engineered ketoreductase (e.g.,such as SEQ ID NO:6): 46→R; 96→V; 99→E; 108→H; 147→M; 152→N; 153→V;157→S; 195→M; 196→L; 199→V,E,R; 206→W; 226→V; 233→G; 245→I; and 249→W.

In some embodiments, the ketoreductase polypeptides of the invention cancomprise a region having an amino acid sequence that is at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a reference sequence based on SEQ ID NO:2, 4 or 60, ora region or domain thereof, such as residues 90-211, with the provisothat the residue corresponding to position 94 is aspartic acid; theresidue corresponding to position 145 is phenylalanine, tyrosine orleucine, particularly phenylalanine or leucine; the residuecorresponding to position 190 is cysteine or proline, particularlyproline; the residue corresponding to position 40 is arginine; theresidue corresponding to position 147 is methionine, and wherein thepolypeptide can additionally have one or more of the followingsubstitutions such that the polypeptide is further improved (e.g., withrespect to stereoselectivity, enzymatic activity, and/orthermostability) over the wild-type kefir ketoreductase or anotherengineered ketoreductase (such as SEQ ID NO:6): 46→R; 96→V; 108→H;152→N; 153→V; 157→S; 195→M; 196→L; 199→V,E,R; 206→W; 226→V; 233→G;245→I; and 249→W.

In some embodiments, the improved engineered ketoreductase enzymescomprise deletions of the naturally occurring ketoreductase polypeptidesas well as deletions of other improved ketoreductase polypeptides. Insome embodiments, each of the improved engineered ketoreductase enzymesdescribed herein can comprise deletions of the polypeptides describedherein. Thus, for each and every embodiment of the ketoreductasepolypeptides of the disclosure, the deletions, which can be internal orexternal (i.e., C- and/or N-terminal truncations) deletions, cancomprise one or more amino acids, 2 or more amino acids, 3 or more aminoacids, 4 or more amino acids, 5 or more amino acids, 6 or more aminoacids, 8 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, up to 10% of the total number of amino acids, up to 20% of thetotal number of amino acids, or up to 30% of the total number of aminoacids of the ketoreductase polypeptides, as long as the functionalactivity of the ketoreductase activity is maintained. In someembodiments, the deletions can comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-25, 1-30, 1-35 or about 1-40 amino acid residues. In some embodiments,the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 amino acids. In someembodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 amino acid residues.

The present invention also provides fragments of the above-describedimproved engineered ketoreductase enzymes that exhibit ketoreductaseactivity where the amino acid sequence of the fragment comprises thefollowing features: (a) the residue corresponding to residue X94 isaspartic acid, (b) the residue corresponding to residue X145 istyrosine, tryptophan, phenylalanine, or leucine, particularlyphenylalanine or leucine; and (c) the residue corresponding to residueX190 is a cysteine or proline, where amino acid position is determinedby optimal alignment with a reference sequence selected from SEQ ID NO:2, 4, or 60.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-stereomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys(nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

The residues in the catalytic domain of L. kefir are 5143, Y156, K160,and N114. The binding pocket domain residues are located at positions94, 96, 153, 150, 144, 145, 190, 195, 196, 199, 202, 206, 211, and 249in L. kefir KRED. Q252 is located in the Mg2⁺ binding domain. Residuesin the NADP binding domain are residues 14-20, 37-40, 62-64, 90-93, 113,141, 188-191, 193, and 195. Sequence-activity analyses indicated thatthe specific substitutions described herein at positions 94, 145, and190 were important with respect to the ability of the engineeredketoreductase polypeptides described herein to stereoselectively reducethe substrate N-methyl-3-keto-3-(2-thienyl)-1-propanamine to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine at a high percentstereomeric excess. These positions directly interact with the substratein the binding pocket of the ketoreductase.

Polynucleotides Encoding Engineered Ketoreductases

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductase enzymes. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered ketoreductase canbe introduced into appropriate host cells to express the correspondingketoreductase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved ketoreductaseenzymes disclosed herein. Thus, having identified a particular aminoacid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in Table 2. In various embodiments, the codons arepreferably selected to fit the host cell in which the protein is beingproduced. For example, preferred codons used in bacteria are used toexpress the gene in bacteria; preferred codons used in yeast are usedfor expression in yeast; and preferred codons used in mammals are usedfor expression in mammalian cells. By way of example, the polynucleotideof SEQ ID NO: 1 has been codon optimized for expression in E. coli, butotherwise encodes the naturally occurring ketoreductase of Lactobacilluskefir.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a ketoreductase polypeptide with an amino acid sequence thathas at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more sequence identity to any of thereference engineered ketoreductase polypeptides described herein, wherethe encoded ketoreductase polypeptide comprises an amino acid sequencethat has at least the following features: (1) the amino acid residuecorresponding to residue position 94 of SEQ ID NO:2, 4 or 60 is anacidic amino acid, (2) the amino acid residue corresponding to residueposition 145 of SEQ ID NO:2, 4 or 60 is an aromatic amino acid orleucine, and (3) the amino acid residue corresponding to residueposition 190 of SEQ ID NO:2, 4 or 60 is a cysteine or a constrainedamino acid. In some embodiments, the polynucleotides with the specifiedsequence identity above encode ketoreductase polypeptides having atleast the following features: (1) the amino acid residue correspondingto position 94 is aspartic acid, (2) the amino acid residuecorresponding to position 145 is tyrosine, tryptophan, phenylalanine, orleucine, particularly phenylalanine or leucine, and (3) the amino acidresidue corresponding to position 190 is cysteine or proline. In someembodiments, the polynucleotides with the above specified sequenceidentity encode ketoreductase polypeptides having at least the followingfeatures: (1) the amino acid residue corresponding to position 94 isaspartic acid, (2) the amino acid residue corresponding to position 145is phenylalanine or leucine, and (3) the amino acid corresponding toposition 190 is cysteine or proline, particularly proline. In someembodiments, the polynucleotides encode an engineered ketoreductasepolypeptide comprising an amino acid sequence selected from SEQ ID NOS:SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

In some embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NO: 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, and 31. In some embodiments, the polynucleotides arecapable of hybridizing under highly stringent conditions to apolynucleotide selected from SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, and 31, where the polynucleotide that hybridizes underhighly stringent conditions encode a functional ketoreductase capable ofconverting the substrate of structural formula (I) to the product ofstructural formula (II), including, for example, the substrate havingthe structural formula (III) to the product having the structuralformula (IV).

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more sequence identity at the nucleotide level to a referencepolynucleotide encoding the engineered ketoreductase. In someembodiments, the reference polynucleotide is selected frompolynucleotide sequences represented by SEQ ID NO: 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, and 31.

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006, which are incorporated herein by reference.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, trp operon, Streptomycescoelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene(sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (See, Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. USA 75: 3727-3731, which is incorporated herein byreference), as well as the tac promoter (DeBoer et al., 1983, Proc.Natl. Acad. Sci. USA 80: 21-25, which is incorporated herein byreference). In some embodiments, the promoters can be those based onbacteriophage promoters, such as phage λ promoters. Further promotersare described in Sambrook et al., supra, which is incorporated herein byreference.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GALL), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488, which is incorporated herein by reference.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra, which isincorporated herein by reference.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990, which is incorporated hereinby reference.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137, whichis incorporated herein by reference.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra, which is incorporated herein by reference.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (See, WO 95/33836, which is incorporated herein by reference).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the KRED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered ketoreductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori (as shown in the plasmid of FIG. 5) or the origins of replication ofplasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), orpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,or pAM 1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes it's functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad. Sci. USA75:1433, which is incorporated herein by reference).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTM™ expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, La Jolla Calif., andplasmids which are derived from pBR322 (Gibco BRL), pUC (Gibco BRL),pREP4, pCEP4 (Invitrogen) or pPoly (See Lathe et al., 1987, Gene57:193-201, which is incorporated herein by reference).

Host Cells for Expression of Ketoreductase Polypeptides

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase enzyme in the host cell. Host cells for use inexpressing the KRED polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Lactobacillus kefir,Lactobacillus brevis, Lactobacillus minor, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the ketoreductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

As described in Example 1, an exemplary host cell is Escherichia coliW3110. In Example 1, the expression vector was created by operativelylinking a polynucleotide encoding an improved ketoreductase into theplasmid pCK110900 operatively linked to the lac promoter under controlof the lad repressor. The expression vector also contained the P15aorigin of replication and the chloramphenicol resistance gene. Cellscontaining the subject polynucleotide in Escherichia coli W3110 wereisolated by subjecting the cells to chloramphenicol selection.

Methods of Generating Engineered Ketoreductase Polypeptides.

In some embodiments, to make the improved KRED polynucleotides andpolypeptides of the present disclosure, the naturally-occurringketoreductase enzyme that catalyzes the reduction reaction is obtained(or derived) from Lactobacillus kefir or Lactobacillus brevis orLactobacillus minor. In some embodiments, the parent polynucleotidesequence is codon optimized to enhance expression of the ketoreductasein a specified host cell. As an illustration, the parentalpolynucleotide sequence encoding the wild-type KRED polypeptide ofLactobacillus kefir was constructed from oligonucleotides prepared basedupon the known polypeptide sequence of Lactobacillus kefir KRED sequenceavailable in Genbank database (Genbank accession no. AAP94029GI:33112056). The parental polynucleotide sequence, designated as SEQ IDNO: 1, was codon optimized for expression in E. coli and thecodon-optimized polynucleotide cloned into an expression vector, placingthe expression of the ketoreductase gene under the control of the lacpromoter and lad repressor gene. Clones expressing the activeketoreductase in E. coli were identified and the genes sequenced toconfirm their identity. The sequence designated (SEQ ID NO:1) was theparent sequence utilized as the starting point for most experiments andlibrary construction of engineered ketoreductases evolved from theLactobacillus kefir ketoreductase.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods, as discussed above. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529). All of which references areincorporated herein by reference.

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD⁺ or NADP⁺. In this reaction,the NADH or NADPH is consumed (oxidized) by the ketoreductase as theketoreductase reduces a ketone substrate to the corresponding hydroxylgroup. The rate of decrease of NADH or NADPH concentration, as measuredby the decrease in absorbance or fluorescence, per unit time indicatesthe relative (enzymatic) activity of the KRED polypeptide in a fixedamount of the lysate (or a lyophilized powder made therefrom). Where theimproved enzyme property desired is thermal stability, enzyme activitymay be measured after subjecting the enzyme preparations to a definedtemperature and measuring the amount of enzyme activity remaining afterheat treatments. Clones containing a polynucleotide encoding aketoreductase are then isolated, sequenced to identify the nucleotidesequence changes (if any), and used to express the enzyme in a hostcell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Midland Certified Reagent Company, Midland, Tex.,The Great American Gene Company, Ramona, Calif., ExpressGen Inc.Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a compound. The compound may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette Guerin) andCorynebacterium parvum.

Methods of Using the Engineered Ketoreductase Enzymes and CompoundsPrepared Therewith

In some embodiments, the invention provides a method for producing an(S)-3-aryl-3-hydroxypropanamine, the method comprising:

(a) providing a 3-aryl-3-ketopropanamine substrate having the structureof formula (I):

(b) contacting or incubating the 3-aryl-3-ketopropanamine substrate withone (or more) of the ketoreductase polypeptides described herein in areaction mixture under conditions suitable for reduction or conversionof the substrate to an (S)3-aryl-3-hydroxypropanime product having thestructural formula (II):

wherein for (I) and (II), R₁ and R₂ are each independently selected fromthe group consisting of hydrogen, an optionally substituted lower alkyl,an optionally substituted cycloalkyl, an optionally substituted aryl, oralternatively, where R₁ and R₂ together form an optionally substitutedcycloalkyl or an optionally substituted cycloaryl having 3-7 carbonatoms; R₃, R₄, R₅, and R₆ are each independently selected from the groupconsisting of hydrogen and an optionally substituted lower alkyl; and R₇is an optionally substituted aryl.

The resulting (S)-3-aryl-3-hydroxypropanamine may be recovered from step(b) and optionally purified using known methods.

Typically, for each of (I) and (II), R₁ and R₂ are each independentlyhydrogen, a lower alkyl of from one to ten or one to six carbon atoms,or a phenyl. Usually, one of R₁ and R₂ is hydrogen and the other ismethyl or both R₁ and R₂ are methyl. Each of R₃, R₄, R₅, and R₆ istypically hydrogen or a lower alkyl of one to six carbon atoms, and moretypically each of R₃, R₄, R₅, and R₆ is hydrogen. R₇ is typically anoptionally substituted thiophenyl (such as, for example, an optionallysubstituted 2-thienyl or an optionally 3-thienyl) or an optionallysubstituted phenyl. More typically, R₇ is 2-thienyl or phenyl. Usually,R₇ is 2-thienyl. In certain exemplary substrates/products, R₁ ishydrogen, R₂ is methyl, R₃-R₆ are each hydrogen, R₇ is selected from thegroup consisting of 2-thienyl and phenyl; and R₁ and R₂ are methyl,R₃-R₆ are each hydrogen, R₇ is selected from the group consisting of2-thienyl and phenyl.

In some embodiments, any one of the ketoreductase polypeptides providedherein can be used in the production of intermediates for the synthesisof Duloxetine (i.e.,(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1 amine), adrug for the treatment of depression. In certain approaches to thesynthesis of Duloxetine, an important step is the conversion of certaincompounds of formula (I) to the corresponding compounds of formula (II).

More specifically, the ketoreductase enzymes described herein cancatalyze the reduction of the substrate compound of structural formula(III), N-methyl-3-keto-3-(2-thienyl)-1-propanamine (“the monomethylsubstrate”, i.e., with respect to formula (I), one of R₁ and R₂ ishydrogen and the other is methyl, R₃, R₄, R₅, and R₆ are each hydrogen,and R₇ is 2-thienyl):

to the corresponding stereoisomeric alcohol product of structuralformula (IV), (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“themonomethyl product””, i.e., with respect to formula (II), one of R₁ andR₂ is hydrogen and the other is methyl, R₃, R₄, R₅, and R₆ are eachhydrogen, and R₇ is 2-thienyl):

The ketoreductase polypeptides described herein are also useful forcatalyzing the reduction of the substrate compound of structural formula(V), N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (“the dimethylsubstrate”, i.e., with respect to formula (I), R₁ and R₂ are eachmethyl, R₃, R₄, R₅, and R₆ are each hydrogen, and R₇ is 2-thienyl):

to the stereoisomeric alcohol product of structural formula (VI),(S)—N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“the dimethylproduct”, i.e., with respect to formula (II), R₁ and R₂ are each methyl,R₃, R₄, R₅, and R₆ are each hydrogen, and R₇ is 2-thienyl):

Products (IV) and (VI) are both useful as intermediates in the synthesisof Duloxetine.

For instance, in some embodiments of this method for reducing thesubstrate, i.e. the monomethyl substrate) to the corresponding product,the ketoreductase polypeptide useful in the method has, as compared tothe wild-type L. kefir or L. brevis or L. minor KRED sequences of SEQ IDNO: 2, 4 or 60, an amino acid sequence with at least the followingfeatures: (1) residue corresponding to X94 is an acidic residue, (2)residue corresponding to X145 is an aromatic residue or leucine, and (3)residue corresponding to X190 is a cysteine or a constrained residue. Insome embodiments, the ketoreductase polypeptide has, as compared to thewild-type L. kefir or L. brevis or L. minor KRED sequences of SEQ IDNO:2, 4 or 60, at least the following features: (1) residue 40 is anaspartic acid, (2) residue 190 is a phenylalanine or leucine, and (3)residue corresponding to X190 is a cysteine or proline. As noted herein,the polynucleotides can encode a ketoreductase polypeptide with theforegoing features, and have in addition, one or more mutations at otheramino acid residues as compared to the references sequences of SEQ IDNO:2, 4, or 60.

In some embodiments of this method for reducing the monomethyl substrateto the monomethyl product, the substrate is reduced to the product ingreater than about 99% stereomeric excess, wherein the ketoreductasepolypeptide comprises a sequence that corresponds to SEQ ID NO: 8, 10,12, 14, 16, 18, 20, or 22.

In some embodiments of this method for reducing the monomethyl substrateto the corresponding monomethyl product, at least about 95% of thesubstrate is converted to the product in less than about 24 hours whencarried out with greater than about 100 g/L of substrate and less thanabout 5 g/L of the polypeptide, wherein the polypeptide comprises anamino acid sequence corresponding to SEQ ID NO: 24, 26, 28, or 32.

In some embodiments of this method for reducing the monomethyl substrateto the product, at least about 10-20% of 1 g/L substrate is converted tothe corresponding monomethyl product in less than about 24 hours withabout 10 g/L of the polypeptide, wherein the polypeptide comprises anamino acid sequence corresponding to 12, 14, 16, 18, 22, 24, 26, 28, 30,or 32.

In some embodiments, the method for catalyzing the reduction of the3-aryl-3-ketopropanamine substrate compound of structural formula (I),to the steroisomeric alcohol product of structural formula (II)comprises contacting the ketoreductase polypeptide under reactionconditions in which the pH is about 8 or below. In some embodiments, thereaction condition is a pH from about 4 to 8. In some embodiments, thereaction condition is a pH of about 6 to 8. In some embodiments, thereaction condition is a pH of about 6.5.

In some embodiments, the reduction of the substrate compound ofstructural formula (I) to the corresponding alcohol product ofstructural formula (II) is carried out in the presence of isopropylalcohol (IPA). In some embodiments, the IPA is present at ≧50% v/v. Insome embodiments, the IPA in the reaction is present at least about 75%v/v. In some embodiments, the IPA is present at least about 90% v/v. Insome embodiments, the reaction condition is from about pH 9 to about 10and has ≧75% v/v of IPA.

In some embodiments where the substrate isN,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine, the reaction conditionis chosen to reduce or minimize the formation of side products, such as1-(thiophen-2-yl)prop-2-en-1-one); 1 (thiophen-2-yl)propan-1-one;1-(thiophen-2-yl)propan-1-ol; and 1-(thiophen-2-yl)prop-2-en-1-ol.

As is known by those of skill in the art, ketoreductase-catalyzedreduction reactions typically require a cofactor. Reduction reactionscatalyzed by the engineered ketoreductase enzymes described herein alsotypically require a cofactor, although many embodiments of theengineered ketoreductases require far less cofactor than reactionscatalyzed with wild-type ketoreductase enzymes. As used herein, the term“cofactor” refers to a non-protein compound that operates in combinationwith a ketoreductase enzyme. Cofactors suitable for use with theengineered ketoreductase enzymes described herein include, but are notlimited to, NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the reaction mixture. The reduced NAD(P)H form canbe optionally regenerated from the oxidized NAD(P)⁺ form using acofactor regeneration system.

The term “cofactor regeneration system” refers to a set of reactantsthat participate in a reaction that reduces the oxidized form of thecofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD⁺ or NADP⁺, respectively, are known in the art and may be usedin the methods described herein.

The terms “secondary alcohol dehydrogenase” and “sADH” are usedinterchangeably herein to refer to an NAD⁺ or NADP⁺-dependent enzymethat catalyzes the conversion of a secondary alcohol and NAD⁺ or NADP⁺to a ketone and NADH or NADPH, respectively. Scheme (1), below,describes the reduction of NAD⁺ or NADP⁺ by a secondary alcohol,illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the ketoreductase-catalyzed reduction reactionsdescribed herein include both naturally occurring secondary alcoholdehydrogenases, as well as non-naturally occurring secondary alcoholdehydrogenases. Naturally occurring secondary alcohol dehydrogenasesinclude known alcohol dehydrogenases from, Thermoanaerobium brockii,Rhodococcus erythropolis, Lactobacillus kefir, Lactobacillus minor andLactobacillus brevis, and non-naturally occurring secondary alcoholdehydrogenases include engineered alcohol dehydrogenases derivedtherefrom. Secondary alcohol dehydrogenases employed in the methodsdescribed herein, whether naturally occurring or non-naturallyoccurring, may exhibit an activity of at least about 1 μmol/min/mg,sometimes at least about 10 μmol/min/mg, or at least about 10²μmol/min/mg, up to about 10³ μmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In some embodiments, the secondaryalcohol is isopropanol. Suitable aryl-alkyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, the resulting NAD⁺ orNADP⁺ is reduced by the coupled oxidation of the secondary alcohol tothe ketone by the secondary alcohol dehydrogenase. Some engineeredketoreductases also have activity to dehydrogenate a secondary alcoholreductant. In some embodiments using secondary alcohol as reductant, theengineered ketoreductase and the secondary alcohol dehydrogenase are thesame enzyme.

Accumulation of acetone generated by reduction of isopropanol by theKRED enzymes of the invention can prevent the desired reaction(reduction of N-methyl-3-keto-3-(2-thienyl)-1-propanamine to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine) from going tocompletion. Accordingly, in certain embodiments, the desired reductionreaction is carried out at reduced pressure (e.g. at 100 Torr) to removeacetone from the reaction mixture by distillation. In such embodiments,isopropanol can be added to the ongoing reaction to replenish thatconsumed by reduction to acetone as well as that lost by distillationunder reduced pressure.

Suitable exemplary cofactor regeneration systems that may be employedmay include, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acid and NADH orNADPH, respectively. Equation (1), below, describes the glucosedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by glucose:

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference. FIG. 1 depicts the conversion of the substrate compound offormula (III) to the corresponding product of formula (IV) usingcofactors NAD(P)H/NAD(P)⁺ and a glucose/glucose dehydrogenase cofactorrecycling system.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring, may be readily identified usingthe assay described in Example 4 of PCT publication WO 2005/018579, thedisclosure of which is incorporated herein by reference. Exemplarynon-naturally occurring glucose dehydrogenases are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 62, 64, 66, 68, 122, 124, and126. The polynucleotide sequences that encode them are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 61, 63, 65, 67, 121, 123, and125, respectively. All of these sequences are incorporated herein byreference. Additional non-naturally occurring glucose dehydrogenasesthat are suitable for use in the ketoreductase-catalyzed reductionreactions disclosed herein are provided in U.S. application publicationNos. 2005/0095619 and 2005/0153417, the disclosures of which areincorporated herein by reference.

Glucose dehydrogenases employed in the ketoreductase-catalyzed reductionreactions described herein may exhibit an activity of at least about 10μmol/min/mg and sometimes at least about 10² μmol/min/mg or about 10³μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in the assaydescribed in Example 4 of PCT publication WO 2005/018579.

The ketoreductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not completelyinactivate the ketoreductase enzyme. Appropriate co-solvent systems canbe readily identified by measuring the enzymatic activity of thespecified engineered ketoreductase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. Generally, the reduction can be carried outat a pH of about 7 or below, usually in the range of from about 3 toabout 7. In some embodiments, the reduction is carried out at a pH ofabout 6 or below, usually in the range of from about 4 to about 6. Insome embodiments, the reduction is carried out at a pH of about 6 orbelow, often in the range of from about 5 to about 6. In a specificembodiment, the reduction may be carried out at pH 6.

During the course of the reduction reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range by the addition of an acid ora base during the course of the reaction. Alternatively, the pH may becontrolled by using an aqueous solvent that comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,for example, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used.

When a glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (3) can cause the pH of the reaction mixture to drop if theresulting aqueous gluconic acid is not otherwise neutralized. The pH ofthe reaction mixture may be maintained at the desired level by standardbuffering techniques, wherein the buffer neutralizes the gluconic acidup to the buffering capacity provided, or by the addition of a baseconcurrent with the course of the conversion. Combinations of bufferingand base addition may also be used. Suitable buffers to maintain desiredpH ranges are described above. Suitable bases for neutralization ofgluconic acid are organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like. The addition of abase concurrent with the course of the conversion may be done manuallywhile monitoring the reaction mixture pH or, more conveniently, by usingan automatic titrator as a pH stat. A combination of partial bufferingcapacity and base addition can also be used for process control.

When base addition is employed to neutralize gluconic acid releasedduring a ketoreductase-catalyzed reduction reaction, the progress of theconversion may be monitored by the amount of base added to maintain thepH. Typically, bases added to unbuffered or partially buffered reactionmixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, the a co-factor regenerating system may comprise aformate dehydrogenase. The terms “formate dehydrogenase” and “FDH” areused interchangeably herein to refer to an NAD⁺ or NADP⁺-dependentenzyme that catalyzes the conversion of formate and NAD⁺ or NADP⁺ tocarbon dioxide and NADH or NADPH, respectively. Formate dehydrogenasesthat may be suitable for use as cofactor regenerating systems in theketoreductase-catalyzed reduction reactions described herein includeboth naturally occurring formate dehydrogenases, as well asnon-naturally occurring formate dehydrogenases. Formate dehydrogenasesinclude those corresponding to SEQ ID NOS: 70 (Pseudomonas sp.) and 72(Candida boidinii) of PCT publication WO 2005/018579, which are encodedby polynucleotide sequences corresponding to SEQ ID NOS: 69 and 71,respectively, of PCT publication 2005/018579, the disclosures of whichare incorporated herein by reference. Formate dehydrogenases that may beemployed in the methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher, and can bereadily screened for activity in the assay described in Example 4 of PCTpublication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂ ⁻),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, HCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa (pKa=3.7in water) formate is present as both HCO₂ ⁻ and HCO₂H in equilibriumconcentrations. At pH values above about pH 4, formate is predominantlypresent as HCO₂ ⁻. When formate is provided as formic acid, the reactionmixture is typically buffered or made less acidic by adding a base toprovide the desired pH, typically of about pH 5 or above. Suitable basesfor neutralization of formic acid include, but are not limited to,organic bases, for example amines, alkoxides and the like, and inorganicbases, for example, hydroxide salts (e.g., NaOH), carbonate salts (e.g.,NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basic phosphate salts (e.g.,K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂ ⁻, Equation (2) below, describes the formatedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by formate:

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the pH of the reaction mixture may be maintained atthe desired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions.

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the ketoreductase substrate.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

In carrying out the stereoselective reduction reactions describedherein, the engineered ketoreductase enzyme, and any enzymes comprisingthe optional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the engineered ketoreductase enzymeand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. For example,in some embodiments one set of host cells can be transformed withgene(s) encoding the engineered ketoreductase enzyme and another set canbe transformed with gene(s) encoding the cofactor regeneration enzymes.Both sets of transformed cells can be utilized together in the reactionmixture in the form of whole cells, or in the form of lysates orextracts derived therefrom. In other embodiments, a host cell can betransformed with gene(s) encoding both the engineered ketoreductaseenzyme and the cofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineeredketoreductase enzyme and/or the optional cofactor regeneration enzymes,or cell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of ketoreductase substrate employed. Thefollowing guidelines can be used to determine the amounts ofketoreductase, cofactor, and optional cofactor regeneration system touse. Generally, keto substrates can be employed at a concentration ofabout 20 to 300 grams/liter using from about 50 mg to about 5 g ofketoreductase and about 10 mg to about 150 mg of cofactor. Those havingordinary skill in the art will readily understand how to vary thesequantities to tailor them to the desired level of productivity and scaleof production. Appropriate quantities of optional cofactor regenerationsystem may be readily determined by routine experimentation based on theamount of cofactor and/or ketoreductase utilized. In general, thereductant (e.g., glucose, formate, isopropanol) is utilized at levelsabove the equimolar level of ketoreductase substrate to achieveessentially complete or near complete conversion of the ketoreductasesubstrate.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, ketoreductase, and ketoreductase substrate may be added firstto the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, ketoreductase, and cofactor maybe added and mixed into the aqueous phase first. The organic phase maythen be added and mixed in, followed by addition of the ketoreductasesubstrate. Alternatively, the ketoreductase substrate may be premixed inthe organic phase, prior to addition to the aqueous phase

Suitable conditions for carrying out the ketoreductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredketoreductase enzyme and substrate at an experimental pH and temperatureand detecting product, for example, using the methods described in theExamples provided herein.

The ketoreductase catalyzed reduction is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or near complete, reduction of substrate is obtained.Reduction of substrate to product can be monitored using known methodsby detecting substrate and/or product. Suitable methods include gaschromatography, HPLC, and the like. Conversion yields of the alcoholreduction product generated in the reaction mixture are generallygreater than about 50%, may also be greater than about 60%, may also begreater than about 70%, may also be greater than about 80%, may also begreater than 90%, and are often greater than about 97%.

The present invention provides a method for making an(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine, the method comprising:

(a) providing a 3-aryl-3-ketopropanamine substrate having the structureof formula (I):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl;(b) contacting or incubating the 3-aryl-3-ketopropanamine substrate withone or more ketoreductase polypeptides of the present invention in areaction mixture under conditions suitable for reduction or conversionof the substrate to an (S)-3-aryl-3-hydroxypropanamine product havingthe structural formula (II):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl;(c) demethylating the (S)-3-aryl-3-hydroxypropanamine (i.e.,N,N-dimethyl-3-hydroxy-3-(aryl)-propanamine) product of step (b) in areaction mixture under conditions suitable for producing an(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine having the formula ofstructure (II):

wherein one of R₁ and R₂ is methyl and the other is hydrogen, R₃, R₄,R₅, and R₆ are each independently selected from the group consisting ofhydrogen and a an optionally substituted lower alkyl, and R₇ is anoptionally substituted aryl.

The substituents R₃-R₇ are as described hereinabove for compounds havingthe structures of formula (I) and (II). Typically, R₃, R₄, R₅, and R₆are all hydrogen and R₇ is an aryl selected from the group consisting ofphenyl and a thienyl. Usually, R₇ is 2-thienyl.

Conditions suitable for producing the(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine in step (b) includecontacting the N,N-dimethyl-3-hydroxy-3-(aryl)-propanamine product ofstep (b) with a demethylating agent in the presence of a base to form anintermediate that is subjected to further hydrolysis to yield the(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine product of step (c). Theterm “demethylating agent” refers herein to a compound that facilitatesremoval of a methyl group from an amine. Suitable demethylating agentsinclude chloroformate and derivatives thereof, phosgene and derivativesthereof, and other suitable compounds that are well known in the art.Exemplary chloroformate derivatives include ethyl chloroformate, methylchloroformate, propyl chloroformate, butyl chloroformate, i-butylchloroformate, phenyl chloroformate, 2,2,2-trichloroethyl chloroformate,2-chloroethyl chloroformate, 2-iodoethyl chloroformate, benzylchloroformate, nitrobenzyl chloroformate, 1-chloroethyl chloroformate,2,2-dichloroethyl chloroformate, 1,1-dimethyl-2,2,2-trichloroethylchloroformate, 1,1-dimethyl-2-chloroethyl chloroformate,1,1-dimethyl-2-bromoethyl chloroformate, and the like. Typically, thechloroformate derivative is i-butyl chloroformate or phenylchloroformate. Phosgene derivatives suitable for use in the practice ofthe invention include diphosgene (i.e., trichloromethyl chloroformate),triphosgene (i.e., bis(trichloromethyl carbonate), and the like.

Suitable bases to use in conjunction with the demethylating agentinclude amines (e.g., trialkylamines such as triethylamine,trimethylamine, dialkylamines, and the like), hydroxides of an alkalimetal or their salts with weak acid (such as, for example, sodiumhydroxide, potassium hydroxide, sodium carbonate, sodiumhydrogencarbonate, potassium carbonate, potassium hydrogencarbonate,cesium carbonate, tripotassium phosphate, tripotassium phosphatedehydrate, and the like), hydroxides of quaternary ammonium or theirsalts with a weak acid, and the like. Typically the base is an amine,such as, for example, triethylamine.

The intermediate is prepared in a reaction mixture of the demethylatingagent, base, and a solvent, such as, for example, a pyrrolidone, aketone (e.g., acetone, ethyl methyl ketone, and the like), a dipolaraprotic solvent (e.g., dimethyl sulfoxide (DMSO),N-methyl-2-pyrrolidone, dimethylformamide, dimethylacetamide, and thelike), an aromatic hydrocarbon (e.g., benzene, toluene, xylene,mesitylene, and the like), a nitrile (e.g., acetonitrile, and the like),an ether (e.g., t-butyl methyl ether, diisopropyl ether,tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, anisole, and thelike), an amide and the like, as well as combinations of any two or morethereof. Typically, the solvent is an aromatic hydrocarbon, such as, forexample, toluene. If the solvent is not replaced for the subsequencehydrolysis step, the solvent is typically an aromatic hydrocarbon, suchas, for example, benzene, toluene, xylene, and mesitylene, an ether,such as, for example, t-butyl methyl ether, diisopropyl ether,tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane and anisole, or anycombination of two or more thereof.

The reaction is typically carried out at a temperature in the range offrom about −30° C. to about 100° C. Typically, the reaction is carriedout at a temperature in the range of from about 0° C. to about 90° C.,and usually at a temperature in the range of from about 25° C. or 30° C.to about 80° C., and often at a temperature of about 75° C.

The subsequent hydrolysis reaction is carried out in the presence of abase and a solvent, such as, for example, an alcohol (e.g., methanol,ethanol, propanol, isopropanol, butanol, and the like), an amide, apyrrolidone, a dipolar aprotic solvent (e.g., DMSO,N-methyl-2-pyrrolidone, dimethylformamide, dimethylacetamide, and thelike), a ketone (e.g., acetone, ethyl methyl ketone, and the like), anaromatic hydrocarbon (e.g., benzene, toluene, xylene, mesitylene, andthe like), and ether (e.g., butyl methyl ether, eiisopropyl ether,tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, anisole, and thelike), water, and the like, as well as any combination of two or morethereof. Aqueous co-solvent systems that are suitable for use in thehydrolysis system include water and an alcohol in a mixture of 25:75,50:50, or 75:25 water:alcohol. Bases suitable for using in thehydrolysis step include a hydroxide of an alkali metal or salt thereofwith a weak acid, a hydroxide of quaternary ammonium or salt thereofwith a weak acid, and the like. Exemplary bases include sodiumhydroxide, potassium hydroxide, sodium carbonate, sodiumhydrogencarbonate, potassium carbonate, potassium hydrogencarbonate,cesium carbonate, tripotassium phosphate and tripotassium phosphatedihydrate. Typically, the hydrolysis is carried out using a hydroxide ofan alkali metal, such as, for example, potassium or sodium, in awater-alcohol co-solvent system, such as for example water-methanol,water-isopropanol, and the like. Conditions suitable for carrying outdemethylation are described in U.S. Patent Publication 2006/0167278,U.S. Pat. No. 5,023,269, U.S. Pat. No. 5,491,243, U.S. Pat. No.6,541,668, WO 2007/095200, EP 0 457 559A2, and EP 0 273 658A1, all ofwhich are incorporated herein by reference.

The (S)—N,N-dimethyl-3-hydroxy-3-(aryl)-propanamine may optionally berecovered after carrying out step (b), prior to the demethylation step(c). The demethylated product,(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine, may subsequently berecovered and optionally further purified after carrying out step (c)using methods that are well known in the art. The present inventionincludes (S)—N-methyl-3-hydroxy-3-(aryl)-propanamine produced by themethods described herein.

In a further embodiment, the present invention provides a method ofmaking an (S)-3-aryloxy-3-(aryl)-propanamine, the method comprising:

(a) providing a 3-aryl-3-ketopropanamine having the structure of formula(I):

(b) contacting the 3-aryl-3-ketopropanamine with a ketoreductasepolypeptide of the present invention in a reaction mixture underconditions sufficient to produce an (S)-3-aryl-3-hydroxypropanaminehaving the structure of formula (II):

and(c) contacting the (S)-3-aryl-3-hydroxypropanamine with an activatedaryl compound in a reaction mixture under conditions sufficient toproduce the (S)-3-aryloxy-3-arylpropanamine having the structure offormula (VII)

wherein for (I), (II), and (VII), R₁ and R₂ are each independentlyselected from the group consisting of hydrogen, an optionallysubstituted lower alkyl, an optionally substituted cycloalkyl, anoptionally substituted aryl, or alternatively, where R₁ and R₂ togetherform an optionally substituted cycloalkyl or an optionally substitutedcycloaryl having 3-7 carbon atoms; R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl; and R₇ is an optionally substitutedaryl and additionally, for (VII), Ar is an optionally substituted arylgroup. Typically, R₁-R₇ are substituents as described hereinabove withrespect to formulas (I) and (II). Typically, Ar is an aryl selected fromthe group consisting of 1-naphthyl, phenyl, 4-trifluoromethylphenyl,2-methylphenyl, 2-methoxyphenyl, and 2-thiomethoxyphenyl.

The method may optionally include a demethylation step if R₁ and R₂ ofthe 3-aryl-3-ketopropanamine are both methyl as described hereinabove.The demethylation step may occur before or after step (c). Preferably,the demethylation step occurs before step (c).

As used herein, the term “activated aryl compound” refers to an arylcompound that is substituted with a leaving group. Suitable leavinggroups include halides (such as, for example, fluoro-, chloro-, and thelike), pseudohalides (such as, for example, sulfonates, such astriflate, tosylate, mesylate, and the like), and the like. Reactionconditions sufficient to produce the 3-aryloxy-3-(aryl)-propanaminehaving the structure of formula (VII) include contacting the3-aryl-3-hydroxypropanamine with the activated aryl compound in thepresence of an alkali metal hydride, such as, for example, sodiumhydride, potassium hydride, and the like, or an alkali metal hydroxide,such as, for example, potassium hydroxide, sodium hydroxide, and thelike. The 3-aryl-3-hydroxypropanamine may also be contacted with an arylcompound activated with a pseudohalide (such as, for example, triflate,tosylate, and the like) or a halide in the presence of a transitionmetal catalyst (such as, for example, palladium (Pd), Copper (Cu), andthe like). The reaction is carried out in a solvent, such as, forexample, N,N-dimethylacetamide, dimethylsulfoxide, dimethylformamide,pyridine, and the like. The reaction is typically carried out at atemperature in the range of from about 10° C. up to the refluxtemperature of the solvent used. Methods for preparing a3-aryloxy-3-(aryl)-propanamine from a 3-aryl-3-hydroxypropanamine aredescribed, for example, in, U.S. Pat. No. 5,023,269, U.S. Pat. No.5,491,243, U.S. Pat. No. 6,541,668, WO 2007/095200, EP 0 457 559A2, andEP 0 273 658A1, all of which are incorporated herein by reference.

The methods for making a 3-aryloxy-3-(aryl)-propanamine optionallyinclude the further step of making a pharmaceutically acceptable salt ofthe 3-aryloxy-3-(aryl)-propanamine. Methods for making apharmaceutically acceptable salt are well known in the art. See, forexample, U.S. Pat. No. 5,023,269, U.S. Pat. No. 5,491,243, U.S. Pat. No.6,541,668, WO 2007/095200, EP 0 457 559A2, and EP 0 273 658A1, all ofwhich are incorporated herein by reference. Pharmaceutically acceptableacid addition salts are typically prepared by reacting a3-aryloxy-3-(aryl)-propanamine with an equimolar or excess amount ofacid. Suitable acids include both inorganic and organic acids. Exemplaryinorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric,sulfuric, phosphoric, metaphosphoric, pyrophosphoric, and the like.Exemplary organic acids include aliphatic mono- and dicarboxylic acids,substituted alkanoic acids (e.g., phenyl-substituted,hydroxyl-substituted, and the like), aromatic acids, aliphatic andaromatic sulfonic acids, and the like.

In some embodiments, the methods relate more specifically to use of theketoreductase polypeptides provided herein in the synthesis ofDuloxetine (i.e.,(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine),having the structural formula (VIII):

and salts, hydrates and solvates thereof. Accordingly, in a method forthe synthesis of the compound of formula (VIII), a step in the methodcan comprise stereoselectively reducing the substrate of structuralformula (III) to the corresponding alcohol product of structural formula(IV) by contacting or incubating the substrate with any one or more ofthe ketoreductase polypeptides of the disclosure. Once formed, thecompound of structure (IV) can be used with known methods for synthesisof the compound of formula (VIII). An exemplary process is described inpatent publication US2007/0167636, the description of which areincorporated herein by reference.

The present invention therefore provides methods for making Duloxetine,i.e., (3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine(Formula VIII). In one embodiment the method comprises:

(a) providing N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine;

(b) contacting the N,N-dimethyl-3-keto-3-(2-thienyl)-propanamine with aketoreductase polypeptide of the present invention in a reaction mixtureunder conditions sufficient for producing(S)—N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine;

(c) demethylating the(S)—N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine to produce(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine; and

(d) contacting the (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanaminewith an activated naphthalene in a reaction mixture under conditionssufficient to produce(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine(Duloxetine) having the structure of formula (VIII); and(e) recovering the(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine fromthe reaction mixture.

In another embodiment for making Duloxetine, the method comprises:

(a) providing N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine;

(b) contacting the N-dimethyl-3-keto-3-(2-thienyl)-propanamine with aketoreductase polypeptide of the present invention in a reaction mixtureunder conditions sufficient for producing(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine; and

(c) contacting the (S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanaminewith an activated naphthalene in a reaction mixture under conditionssufficient to produce(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1 amine(Duloxetine) having the structure of formula (VIII); and(d) recovering the(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-amine fromthe reaction mixture.

As used herein, the term “activated naphthalene” refers to an activatedaryl as described above, where the aryl group is 1-naphthyl. Reactionconditions for carrying out the naphthylation and demethylation are aspreviously described above in the description of the method for making a3-aryloxy-3-arylpropanamine from an (S)-3-aryl-3-hydroxypropanamine.Demethylating agents suitable for use in the method for makingDuloxetine from(S)—N,N-dimethyl-3-napthalen-1-yloxy-3-thiophen-2-yl-propan-1-diamineinclude those described hereinabove. Methods for preparing a3-aryloxy-3-arylpropanamine from a 3-aryl-3-hydroxypropanamine aredescribed, for example, in, U.S. Pat. No. 5,023,269, U.S. Pat. No.5,491,243, and U.S. Pat. No. 6,541,668, WO 2007/095200, EP 0 457 449A2,and EP 0 273 658A1, all of which are incorporated herein by reference.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

EXAMPLES Example 1 Wild-Type Ketoreductase Gene Acquisition andConstruction of Expression Vectors

Ketoreductase (KRED) encoding genes are designed for expression in E.coli based on the reported amino acid sequence of the ketoreductase andusing standard codon-optimization methods. Standard codon-optimizationsoftware is reviewed in e.g., ÓPTIMIZER: a web server for optimizing thecodon usage of DNA sequences,” Puigbó et al., Nucleic Acids Res. (2007July); 35 (Seb Server issue): W126-31, Epub 2007 Apr. 16. Genes aresynthesized using oligonucleotides, generally composed of 42nucleotides, which are cloned into the expression vector pCK110900,depicted as FIG. 3 in United States Patent Application Publication20060195947, which is incorporated herein by reference, under thecontrol of a lac promoter. This expression vector also contains the P15aorigin of replication and the chloramphenicol resistance gene. Resultingplasmids are transformed into E. coli W3110 using standard methods.Codon-optimized genes and the encoding polypeptides as well are listedin Tables 2 and 3, and their sequences provided in SEQ ID NO: 5-112.Ketoreductases useful for development of enzymes capable of reducingcompounds of Formula (III) to the compounds of Formula (IV) are providedin Table 3, below.

TABLE 3 Abbreviations, Source and References for Useful KetoreductasesMicroorganism Genbank Polypeptide from which enzyme was Accession GIPolynucleotide SEQ ID Number Ketoreductase originally identified Numbernumber SEQ ID NO or source ADH-CM Candida magnoliae AB036927.1 12657576SEQ ID NO: 33 SEQ ID NO: 34 YDL Saccharomyces cerevisiae NP_010159.16320079 SEQ ID NO: 39 SEQ ID NO: 40 ADH-LB Lactobacillus brevis 1NXQ_A30749782 SEQ ID NO: 3 SEQ ID NO: 4 ADH-RE Rhodococcus erythropolisAAN73270.1 34776951 SEQ ID NO: 5 SEQ ID NO: 6 YGL Saccharomycescerevisiae NP_011476 6321399 SEQ ID NO: 37 SEQ ID NO: 38 YPRSaccharomyces cerevisiae NP_010656.1 6320576 SEQ ID NO: 41 SEQ ID NO: 42GRE Saccharomyces cerevisiae NP_014490.1 6324421 SEQ ID NO: 43 SEQ IDNO: 44 ADH-LK Lactobacillus kefir AAP94029.1 33112056 SEQ ID NO: 1 SEQID NO: 2 ADH-SB Sporobolomyces salmonicolor Q9UUN9 30315955 SEQ ID NO:47 SEQ ID NO: 48 ADH-SC Streptomyces coelicolor NP_631415.1 21225636 SEQID NO: 45 SEQ ID NO: 46 ADH-TB Thermoanaerobium brockii X64841.1 1771790SEQ ID NO: 55 SEQ ID NO: 56 ADH-CP Candida parapsilosis BAA24528 2815409Julich Chiral Solutions Cat. No. 03.11 DR-LB Lactobacillus brevisABJ63353.1 116098204 Julich Chiral diacetyl reductase Solutions Cat. No.8.1 ADH-HE Horse liver DEHOAL 625197 SEQ ID NO: 57 SEQ ID NO: 58 ADH-CBCandida boidinii CAD66648 28400789 Julich Chiral Solutions Cat. No.02.10 LDH-LL Lactobacillus leichmannii Fluka Cat. No. 61306 ADH-AFAspergillus flavus P41747 1168346 SEQ ID NO: 49 SEQ ID NO: 50 ADH-OO1Oenococcus oeni ZP_00318704.1 48864831 SEQ ID NO: 51 SEQ ID NO: 52ADH-RU Ralstonia eutropha ZP_00202558.1 46131317 SEQ ID NO: 53 SEQ IDNO: 54As noted above, polynucleotides encoding engineered ketoreductasesdescribed in the present disclosure may also be cloned into vectorpCK110900 for expression in E. coli W3110.

Example 2 Production of Ketoreductase Powders—Shake Flask Procedure

A single microbial colony of E. coli containing a plasmid encoding aketoreductase of interest is inoculated into 50 mL Luria Bertani brothcontaining 30 μg/ml chloramphenicol and 1% glucose. Cells are grownovernight (at least 16 hrs) in an incubator at 30° C. with shaking at250 rpm. The culture is diluted into 250 ml Terrific Broth (12 g/Lbacto-tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/ml chloramphenicol, in a1 liter flask to an optical density at 600 nm (OD₆₀₀) of 0.2 and allowedto grow at 30° C. Expression of the ketoreductase gene is induced byaddition of iso-propyl-β-D-thiogalactoside (“IPTG”) to a finalconcentration of 1 mM when the OD₆₀₀ of the culture is 0.6 to 0.8 andincubation is then continued overnight (at least 16 hrs). Cells areharvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet is resuspended with an equalvolume of cold (4° C.) 100 mM triethanolamine (chloride) buffer, pH 7.0(including 2 mM MgSO₄ in the case of ADH-LK (SEQ ID NO: 4) and ADH-LB(SEQ ID NO: 2) and engineered ketoreductases derived therefrom), andharvested by centrifugation as above. The washed cells are resuspendedin two volumes of the cold triethanolamine (chloride) buffer and passedthrough a French Press twice at 12,000 psi while maintained at 4° C.Cell debris is removed by centrifugation (9000 rpm, 45 min., 4° C.). Theclear lysate supernatant is collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry powder of crudeketoreductase enzyme. Alternatively, the cell pellet (before or afterwashing) may be stored at 4° C. or −80° C.

Example 3 Production of Ketoreductases—Fermentation Procedure

Bench-scale fermentations are carried out at 30° C. in an aerated,agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/L ammoniumsulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogenphosphate trihydrate, 6.25 g/L of potassium dihydrogen phosphate, 6.2g/L of Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, and8.3 ml/L of a trace element solution containing 2 g/L of calciumchloride dihydrate, 2.2 g/L of zinc sulfate septahydrate, 0.5 g/Lmanganese sulfate monohydrate, 1 g/L cuprous sulfate heptahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate).The fermentor is inoculated with a late exponential culture of E. coliW3110 containing a plasmid encoding the ketoreductase gene of interest(grown in a shake flask as described in Example 2) to a starting OD₆₀₀of 0.5 to 2.0. The fermentor is agitated at 500-1500 rpm and air issupplied to the fermentation vessel at 1.0-15.0 L/min to maintain adissolved oxygen level of 30% saturation or greater. The pH of theculture is maintained at 7.0 by addition of 20% v/v ammonium hydroxide.Growth of the culture is maintained by addition of a feed solutioncontaining 500 g/L cerelose, 12 g/L ammonium chloride and 10.4 g/Lmagnesium sulfate heptahydrate. After the culture reaches an OD600 of50, expression of ketoreductase is induced by addition ofisopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mMand fermentation continued for another 14 hours. The culture is thenchilled to 4° C. and maintained at that temperature until harvested.Cells are collected by centrifugation at 5000 G for 40 minutes in aSorval RC12BP centrifuge at 4° C. Harvested cells are used directly inthe following downstream recovery process or they may be stored at 4° C.or frozen at −80° C. until such use.

The cell pellet is resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 6.8, at 4° C. to each volume of wet cell paste.The intracellular ketoreductase is released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate is cooled to 4° C. immediately after disruption. A solutionof 10% w/v polyethyleneimine, pH 7.2, is added to the lysate to a finalconcentration of 0.5% w/v and stirred for 30 minutes. The resultingsuspension is clarified by centrifugation at 5000 G in a standardlaboratory centrifuge for 30 minutes. The clear supernatant is decantedand concentrated ten fold using a cellulose ultrafiltration membranewith a molecular weight cut off of 30 kD. The final concentrate isdispensed into shallow containers, frozen at −20° C. and lyophilized topowder. The ketoreductase powder is stored at −80° C.

Example 4 Analytical Methods: Conversion ofN-methyl-3-keto-3-(2-thienyl)-1-propanamine (“MMAK”) to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-MMAA”)

Achiral HPLC method to determine conversion ofN-methyl-3-keto-3-(2-thienyl)-1-propanamine (“MMAK”) to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-MMAA”):Reduction of MMAK (prepared as described WO2004020391) to (S)-MMAA wasdetermined using an Agilent 1100 HPLC equipped with an Agilent Zorbax 5μm SB-Aq column (15 cm length, 2.1 mm diameter), using 8:2 40 mMNH4Ac/MeCN as eluent at a flow 0.4 ml/min; and a column temperature 50°C. Retention times: MMAA: 1.9 min; MMAK: 2.5 min. The product wasdetermined as the peak area at 235 nm and the substrate as the peak areaat 290 nm.

Chiral HPLC method to determine stereopurity of MMAA: Stereomeric purityof MMAA was determined using an Agilent 1100 HPLC equipped with ShiseidoCD-Ph reverse phase chiral column (25 cm length, 4.6 mm diameter) using7:3 NaClO₄/MeCN as the eluent at a flow rate of 1 ml/min and at atemperature of 35° C.). Retention times: (R)-MMAA: 10.7 min; (S)-MMAA:13.6 min; MMAK: 8.9 min.

Example 5 Prescreen for Ketoreductases Capable of Reducing Isopropanolin the Presence of NADP+ Yielding NADPH and Acetone

Recombinant E. coli colonies carrying a gene encoding ADH-LK or avariant thereof were picked using a Q-Bot® robotic colony picker(Genetix USA, Inc., Boston, Mass.) into 96-well shallow well microtiterplates containing 180 μL, Terrific Broth (TB), 1% glucose and 30 μg/mLchloramphenicol (CAM). Cells were grown overnight at 30° C. with shakingat 200 rpm. A 10 μL, aliquot of this culture was then transferred into96-deep well plates containing 390 μL, Terrific Broth (TB), 1 mM MgSO₄and 30 μg/mL CAM. After incubation of the deep-well plates at 30° C.with shaking at 250 rpm for 2 to 3 hours, recombinant gene expressionwithin the cultured cells was induced by addition of IPTG to a finalconcentration of 1 mM. The plates were then incubated at 30° C. withshaking at 250 rpm for 18 hrs.

Cells were pelleted by centrifugation (4000 RPM, 10 min., 4° C.),resuspended in 400 μL, lysis buffer and lysed by shaking at roomtemperature for 2 hours. The lysis buffer contained 100 mMtriethanolamine (chloride) buffer, pH 7, 1 mg/mL lysozyme, 500 μg/mLpolymixin B sulfate (“PMBS”) and 1 mM MgSO₄. After sealing the plateswith aluminum/polypropylene laminate heat seal tape (Velocity 11 (MenloPark, Calif.), Cat#06643-001), they were shaken vigorously for 2 hoursat room temperature. Cell debris was collected by centrifugation (4000RPM, 10 min., 4° C.) and the clear supernatant was assayed directly orstored at 4° C. until use.

In this assay, 20 μl of sample (diluted in 100 mM triethanolamine(chloride) buffer, at the same pH as the lysis buffer, and 1 mM MgSO₄)was added to 180 μl of an assay mixture in a well of 96-well blackmicrotiter plates. Assay buffer consisted of 100 mM triethanolamine(chloride) buffer, pH 7, 50% isopropyl alcohol (IPA), 1 mM MgSO₄ and 222μM NADP⁺. The reaction was followed by measuring the reduction influorescence of NADP⁺ as it was converted to NADPH using a Flexstation®instrument (Molecular Devices, Sunnyvale, Calif.). NADPH fluorescencewas measured at 445 nm upon excitation at 330 nm. If desired, samples oflysates may be preincubated at 25-40° C. in the presence or absence of50% IPA prior to addition to the assay mixture.

Example 6 High-Throughput Screening for Identification of Variants ofthe Lactobacillus kefer Ketoreductase (ADH-LK) Capable ofStereospecifically ConvertingN-methyl-3-keto-3-(2-thienyl)-1-propanamine (“MMAK”) to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-MMAA”)

The gene encoding ADH-LK, constructed as described in Example 1, wasmutagenized using methods described above and the population of alteredDNA molecules was used to transform a suitable E. coli host strain.Antibiotic resistant transformants were selected and processed toidentify those expressing an ADH-LK variant with an improved ability toreduce N-methyl-3-keto-3-(2-thienyl)-1-propanamine (“MMAK”) (“themonomethyl substrate”) stereospecifically to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (“(S)-MMAA”) (“themonomethyl product”). Cell selection, growth, induction of ADH-LKvariants and collection of cell pellets were as described in Example 5.

Example 7 Assay of ADH-LK Activity Stereospecific Reduction ofN-methyl-3-keto-3-(2-thienyl)-1-propanamine to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine

Cell lysis: Cell pellets were lysed by addition of 400 μL lysis buffer(1 mM MgSO₄, 0.5 mg/ml polymyxin B sulfate (“PMBS”), 1 mg/ml lysozyme,100 mM triethanolamine (pH ˜6), and 1 mg/mL NADP⁺) to each well. Theplates were sealed, shaken vigorously for two hours at room temperature,and then centrifuged at 4000 rpm for 10 minutes at 4° C. Thesupernatants were recovered and stored at 4° C. until use.

Enzymatic reduction reaction: An aliquot (450 μL) of a mixture ofisopropanol and solid substrate(N-methyl-3-keto-3-(2-thienyl)-1-propanamine) was added to each well ofa Costar® deep well plate using a Multidrop instrument (MTX Lab Systems,Vienna Va.), followed by robotic addition of 50 μL of the recoveredlysate supernatant using a Multimek™ instrument (Multimek, Inc., SantaClara Calif.), to provide a reaction comprising 10 mg/ml substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine, 0.1 mg/ml NADP⁺, 10 mMtriethanolamine pH ˜6, and 90% isopropanol (v/v). The plates wereheat-sealed with aluminum/polypropylene laminate heat seal tape(Velocity 11 (Menlo Park, Calif.), Cat#06643-001) at 170° C. for 2.5seconds and then shaken overnight (at least 16 hours) at ambienttemperature. Reactions were quenched by the addition of 1 ml of 95%acetonitrile. Plates were resealed, shaken for 5 min, and thencentrifuged at 4000 rpm for 10 min. A 250 μL aliquot of the clearedreaction mixture was transferred to a new shallow well polypropyleneplate (Costar #3365), which was sealed, after which the extracts weresubjected to HPLC analysis using methods described above (e.g., seeExample 4).

For high throughput screening at pH ˜6 and 85% IPA (v/v), 50 μl of celllysate containing 1 g/L NADP⁺ was transferred to a deep well plate(Costar #3960) containing 450 μl of assay mix (per 100 ml: 5 ml 100 mMtriethanolamine (chloride) (pH 7), 13.4 g MMAK, and 95 ml isopropylalcohol). After sealing the plates, reactions were run for at least 16hours at ambient temperature. Reactions were quenched by the addition of1 ml of 95% acetonitrile, and the plates were sealed withaluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park,Calif.), Cat#06643-001), shaken for 5-10 min, and centrifuged at 4000rpm for 10 minutes. 250 μL of the cleared reaction mixture wastransferred to a new shallow well polypropylene plate (Costar #3365),which was then sealed. The extracts prepared in this manner weresubjected to HPLC analysis as described above.

Variants of the Lactobacillus kefer ketoreductase (ADH-LK) capable ofconverting N-methyl-3-keto-3-(2-thienyl)-1-propanamine to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine were identified usingthe approaches and procedures disclosed above. Multiple iteratations ofthese processes, in which one or more improved isolates from one roundwere used as the starting material for subsequent rounds of mutagenesisand screening, were used to develop or “evolve” Lactobacillus keferketoreductase (ADH-LK) variants with an improved ability to reduceN-methyl-3-keto-3-(2-thienyl)-1-propanamine stereospecifically to(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine.

Example 8 Stereoselective Reduction of MMAK using Isopropyl Alcohol forCo-Factor Regeneration by Engineered Ketoreductases Derived from ADH-LK

Improved ketoreductases derived from ADH-LK variants were evaluated atpreparative scale for the reduction of MMAK as follows. A 100 μLsolution of the ADH-LK variant to be tested (10 mg/mL) and NADP-Na (1mg/mL) in 100 mM triethanolamine (chloride) buffer pH 7 were combined ina 5 mL reaction vial equipped with a magnetic stir bar. Subsequently,850 μL of isopropyl alcohol (“IPA”) were added to the enzyme/NADP-Nasolution, followed by 120 mg of MMAK hydrochloride. The reaction wasstirred at ambient temperature and monitored by HPLC analysis of samplestaken periodically from the reaction using analytical methods disclosedabove (see Example 4). Table 2 provides the SEQ ID NO. corresponding toketoreductase variants, the number of amino acid mutations from thewild-type ADH-LK, and MMAK-reducing activity of each, relative to thatof the enzyme having the amino acid sequence of SEQ ID NO: 6.

This Example illustrates that engineered ketoreductases derived from theketoreductase ADH-LK provide improved activities compared to thewild-type ketoreductase ADH-LK for the reduction of MMAK.

Example 9 Preparative Scale Production of MMAA

A solution of the ketoreductase of SEQ ID NO: 28 (400 mg), and NADP⁺ (20mg) in 10 ml 100 mM triethanolamine (chloride) (pH 8.5) was slowly addedto rapidly-stirring IPA (90 mL) in a 3-neck 250 mL flask equipped with adistillation head, septum and thermoprobe, followed by addition ofsubstrate MMAK (14.6 g). This mixture was vacuum distilled such that thetemperature in the pot is maintained between 23.5 and 25° C. (˜50 mmHg,the vacuum was adjusted to provide the desired reaction temperature).The rate of distillation was about 10 ml/hr. IPA (90% in water was addedperiodically to replace that which was distilled off. At 4 hours, theconversion is 73%. At this time distillation was stopped and anadditional amount of the ketoreductase of SEQ ID NO: 28 (200 mg) andNADP⁺ (10 mg) were added in 5 ml of triethanolamine buffer. Distillationwas continued for a total of 9 hours (conversion: 97%). The reactionmixture was left stirring overnight at room temperature yielding a finalconversion of 99%. Then 30 ml of 25% NaOH were added and, after stirringfor 10 minutes, the lower layer was removed by pipette. The remainingIPA solution was filtered through a celite pad to remove denaturedenzyme and then concentrated using a rotavap. Toluene (70 ml) was addedand the mixture again concentrated. An additional portion of toluene (30ml) was then added and the resulting solution was first warmed toredissolve product that precipitates at room temperature, and thenfiltered through celite. MTBE (40 ml) was added to the filtrate and themixture (containing precipitated product) was stirred overnight. Theslurry was cooled to approximately −10° C. with an ice/methanol bath (1hr) and filtered (MTBE wash). The product was dried in vacuo to affordMMAA as white crystals in ˜75% yield.

All patents, patent publications, journals, and other references citedin this disclosure were hereby incorporated-by-reference in theirentirety.

1. A ketoreductase polypeptide, wherein the polypeptide has an aminoacid sequence that is at least 90% identical to a reference sequence ofSEQ ID NO:2 and has at least the following features: (a) the residuecorresponding to residue X94 is aspartic acid, (b) the residuecorresponding to residue X145 is phenylalanine; and (c) the residuecorresponding to residue X190 is proline.
 2. The ketoreductasepolypeptide of claim 1, wherein the amino acid sequence furthercomprises one or more features selected from the group consisting of:residue corresponding to X40 is a constrained, hydrophilic or basicresidue; residue corresponding to X196 is a non polar or aliphaticresidue; residue corresponding to X226 is a non polar or aliphaticresidue; residue corresponding to X249 is a nonpolar or aromaticresidue; and wherein the amino acid sequence can optionally have one ormore residue differences at other amino acid residues as compared to thereference sequence.
 3. The ketoreductase polypeptide of claim 1, whereinthe polypeptide is capable of converting the substrate to the productwith a percent stereomeric excess of at least 95%.
 4. The ketoreductasepolypeptide of claim 1, wherein the polypeptide is capable of convertingthe substrate to the product with a percent stereomeric excess of atleast 99%.
 5. The ketoreductase polypeptide of claim 1, wherein thepolypeptide is capable of converting the substrate to the product at arate that is at least 10-15 times greater than the rate of conversion ofthe substrate to the product by the reference polypeptide of SEQ IDNO:6.
 6. The ketoreductase polypeptide of claim 1, wherein thepolypeptide is capable of converting the substrate to the product at arate that is at least 15 times greater than the rate of conversion ofthe substrate to the product by the reference polypeptide of SEQ IDNO:6.
 7. The ketoreductase polypeptide of claim 1, wherein thepolypeptide is capable of converting at least 95% of the substrate tothe product in less than about 24 hours when carried out with greaterthan 100 g/L of substrate and less than 5 g/L of the polypeptide.
 8. Theketoreductase polypeptide of claim 1, in which the amino acid sequencefurther comprises one or more of the following features: residuecorresponding to X40 is arginine; residue corresponding to X196 isleucine; residue corresponding to X226 is valine; residue correspondingto X249 is tryptophan; and wherein the amino acid sequence canoptionally have one or more differences at other amino acid residues ascompared to the reference sequence.
 9. A method for producing an(S)-3-aryl-3-hydroxypropanamine, said method comprising: (a) providing a3-aryl-3-ketopropanamine substrate having the structure of formula (I):

(b) contacting the 3-aryl-3-ketopropanamine substrate with theketoreductase polypeptide of claim 1 in a reaction mixture underconditions suitable for reduction or conversion of the substrate to an(S)3-aryl-3-hydroxypropanamine product having the structural formula(II):

wherein for (I) and (II), R₁ and R₂ are each independently selected fromthe group consisting of hydrogen, an optionally substituted lower alkyl,an optionally substituted cycloalkyl, an optionally substituted aryl, oralternatively, wherein R₁ and R₂ together form an optionally substitutedcycloalkyl or an optionally substituted cycloaryl having 3-7 carbonatoms; R₃, R₄, R₅, and R₆ are each independently selected from the groupconsisting of hydrogen and an optionally substituted lower alkyl; and R₇is an optionally substituted aryl.
 10. The method of claim 9, whereinR₃, R₄, R₅, and R₆ are hydrogen, and at least one of R₁ and R₂ ismethyl.
 11. The method of claim 10, wherein R₇ is thienyl.
 12. Themethod of claim 11, wherein the substrate is reduced to product with astereomeric excess of greater than about 99%.
 13. The method of claim 9,wherein the method further comprises a NADH/NADPH cofactor regeneratingsystem.
 14. The method of claim 9, wherein the contacting is carried outat a pH of <8.
 15. The method of claim 9, wherein the contacting is inpresence of at least 50% v/v isopropanol.
 16. The method of claim 9,wherein step (b) is carried out with whole cells that express theketoreductase enzyme, or an extract or lysate of such cells.
 17. Themethod of claim 9, wherein the ketoreductase is isolated and/or purifiedand the reduction reaction is carried out in the presence of a cofactorfor the ketoreductase and optionally a regeneration system for thecofactor.
 18. The method of claim 17, wherein the cofactor regeneratingsystem is selected from the group consisting of glucose dehydrogenaseand glucose, formate dehydrogenase and formate, isopropanol and asecondary alcohol dehydrogenase, and phosphite and phosphitedehydrogenase.
 19. The method of claim 18, wherein the secondary alcoholdehydrogenase is the ketoreductase.
 20. A method of making an(S)—N-methyl-3-hydroxy-3-(aryl)-propanamine, said method comprising: (a)providing a 3-aryl-3-ketopropanamine substrate having the structure offormula (I):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl; (b) contacting the 3-aryl-3-ketopropanamine substrate with one ormore ketoreductase polypeptides of claim 1 in a reaction mixture underconditions suitable for reduction or conversion of the substrate to an(S)-3-aryl-3-hydroxypropanamine product having the structural formula(II):

wherein R₁ and R₂ are each methyl, R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and a anoptionally substituted lower alkyl, and R₇ is an optionally substitutedaryl; (c) demethylating the (S)-3-aryl-3-hydroxypropanamine product ofstep (b) in a reaction mixture under conditions suitable for producingan (S)—N-methyl-3-hydroxy-3-(aryl)-propanamine having the formula ofstructure (II), wherein one of R₁ and R₂ are is methyl and the other ishydrogen, R₃, R₄, R₅, and R₆ are each independently selected from thegroup consisting of hydrogen and an optionally substituted lower alkyl,and R₇ is an optionally substituted aryl.
 21. A method for making a3-aryloxy-3-(aryl)-propanamine, the method comprising: (a) providing a3-aryl-3-ketopropanamine having the structure of formula (I):

(b) contacting the 3-aryl-3-ketopropanamine with a ketoreductasepolypeptide of claim 1 in a reaction mixture under conditions sufficientto produce an (S)-3-aryl-3-hydroxypropanamine having the structure offormula (II):

and (c) contacting the (S)-3-aryl-3-hydropropanamine with an activatedaryl compound in a reaction mixture under conditions sufficient toproduce the (S)-3-aryloxy-3-arylpropanamine having the structure offormula (VII)

wherein for (I), (II), and (VII), R₁ and R₂ are each independentlyselected from the group consisting of hydrogen, an optionallysubstituted lower alkyl, an optionally substituted cycloalkyl, anoptionally substituted aryl, or alternatively, where R₁ and R₂ togetherform an optionally substituted cycloalkyl or an optionally substitutedcycloaryl having 3-7 carbon atoms; R₃, R₄, R₅, and R₆ are eachindependently selected from the group consisting of hydrogen and anoptionally substituted lower alkyl; and R₇ is an optionally substitutedaryl and additionally, for (VII), Ar is an optionally substituted arylgroup.
 22. The method of claim 21, wherein Ar is an aryl selected fromthe group consisting of 1-naphthyl, phenyl, 4-trifluoromethylphenyl,2-methylphenyl, 2-methoxyphenyl, and 2-thiomethoxyphenyl.
 23. A processfor the synthesis of(3S)—N-methyl-3-naphthalen-1-yloxy-3-thiophen-2-yl-propan-1-aminecomprising a step of contacting the substrateN-methyl-3-keto-3-(2-thienyl)-1-propanamine with the ketoreductasepolypeptide of claim 1 in a reaction mixture under conditions suitablefor reduction or conversion of the substrate to product(S)—N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine.